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Introduction

Osteoarthritis (OA) is a primary cause of disability and chronic pain, impacting approximately 500 million people worldwide, with forecasts of almost one billion by 2050.^1,2 OA considerably lowers the quality of life due to its characteristic progressive joint deterioration, pain, and loss of mobility. Though they provide short-term comfort, non-steroidal anti-inflammatory medicines (NSAIDs) and corticosteroids present significant gastrointestinal, cardiovascular, and renal hazards when used long-term.^3,4 While effective for some, surgical interventions are invasive, costly, and not universally accessible.

These restrictions have fueled a rise in interest in integrative and alternative treatments. The monosodium iodoacetate (MIA) model has been a valuable instrument for reproducing important OA characteristics in preclinical research. Therefore, it is a promising avenue for assessing new, non-pharmacologic treatments.^5,6 The GB34 acupoint, located near the fibular head, is often used for joint-related indications in both experimental and clinical settings. Evidence suggests that stimulation at GB34 can modulate inflammatory processes and promote cartilage preservation.^7–9

Acupuncture has gained recognition for its affordability,¹⁰ safety,^11,12 and effectiveness in managing musculoskeletal pain. The American College of Rheumatology conditionally recommends it for knee OA,¹³ with reviews supporting its pain-relieving and functional benefits.^14–17 Acupuncture treatments such as Electroacupuncture (EA), bee venom acupuncture (BVA), and laser acupuncture (LA) differ in mechanism while sharing common targets. Although previous studies have noted the therapeutic benefits of LA, EA, and BVA in OA.^18–23 This study is the first to directly compare these treatments under the same conditions. This direct comparison fills a critical gap in the literature, providing valuable insights for clinical decision-making in integrative pain management. The potential impact of these findings on the future of pain management is significant.

Methods

Ethics Statement

This study was approved by the Dongshin University Animal Committee (DSU-2024-07-04). All animal care and experiments were conducted under the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and Dongshin University Institutional Animal Care and Use Committee policies.

Animals and Osteoarthritis Induction

Forty-eight male Sprague-Dawley rats (8 weeks old, 240–280 g; SAMTAKO Korea) were housed under controlled conditions (22 ± 2°C, reversed 12-hour light/dark cycle) with ad libitum access to food and water.

Male rats were used to reduce differences in pain and inflammatory responses caused by sex hormones. This decision was based on previous research showing that pain from OA varies significantly with a rat’s age and sex.²⁴

OA was induced via intra-articular injection of monosodium iodoacetate (MIA; Sigma-Aldrich, St. Louis, MO). MIA was dissolved in 30 μL of sterile saline at 1 mg. Under brief isoflurane anesthesia, the injection was administered into the medial side of the patellar ligament of both knees using a 19-gauge, 0.5-inch needle, ensuring the needle did not penetrate the cruciate ligaments. The control group received an equivalent volume of sterile saline. Post-injection, the limbs were gently massaged before returning the rats to their housing.

Experimental Groups

Rats were randomly assigned to six groups (n = 8 per group): Control (Con): No OA induction or treatment. OA: MIA-induced OA without treatment. Manual Acupuncture (MA): MIA-induced OA treated with manual acupuncture at GB34. Invasive Laser Acupuncture 830nm (830nm): MIA-induced OA treated with 830nm invasive laser acupuncture at GB34. Electroacupuncture (EA): MIA-induced OA treated with Electroacupuncture at GB34. Bee Venom Acupuncture (BVA): MIA-induced OA treated with bee venom acupuncture at GB34.

Acupuncture Treatments

All acupuncture treatments commenced one-week post-MIA injection and were administered three times per week for four weeks.

Manual Acupuncture (MA): Sterile acupuncture needles (0.25 mm diameter, 13 mm length) were inserted bilaterally at GB34 to a depth of approximately 5 mm. Needles were manually stimulated with gentle twirling for 30 seconds every minute during a 3-minute session.

Invasive Laser Acupuncture 830nm: Using the Ellise device (Wontech Co. Ltd., Daejeon, Republic of Korea), an optic fiber-coupled laser diode was inserted into sterile acupuncture needles. The laser was set at 50Hz, 20mW, and applied for 3 minutes per session at GB34 on both legs to a depth of 5 mm.

Electroacupuncture (EA): Sterile stainless-steel acupuncture needles (0.25 mm diameter, 13 mm length) were inserted bilaterally at GB34 to a depth of approximately 5 mm. Electrical stimulation was applied using a constant current EA device, delivering alternating frequencies of 2/10 Hz at an intensity of 1 mA for 3 minutes per session.

Bee Venom Acupuncture (BVA): A 0.1 mL injection of bee venom solution (1.0 mg/mL) was administered subcutaneously at GB34 using a 30-gauge insulin syringe to a depth of approximately 5 mm.

Behavioral Assessment

Joint pain severity was assessed using the paw withdrawal threshold (PWT) test with manual von Frey filaments. Rats were acclimatized for 10 minutes in individual boxes. A filament with a bending force of 0.6 g was applied perpendicularly to the plantar surface of each hind paw until it bent slightly, and the response was recorded. If no response was observed, a filament with the next higher force was used; if a response occurred, the next lower force was applied. This up-down method continued until a pattern of responses allowed for calculating the 50% withdrawal threshold. Each paw was tested three times, with a 3-minute interval between tests. The mean values were used for statistical analysis. PWT assessments were conducted before MIA induction and weekly thereafter for four weeks.

Behavioral and structural outcomes were assessed up to 4 weeks post-MIA and treatment, a time window known to capture pain progression, early cartilage, and bone alterations in MIA-induced OA models.^25,26

Micro-Computed Tomography (Micro-CT) Imaging

After the treatment period, the rats were euthanized using a carbon dioxide (CO₂) gas chamber via a gradual-fill method, following the AVMA Guidelines for the Euthanasia of Animals (2020 Edition). This approach ensured a humane endpoint, minimizing pain and distress. The right knee joints were harvested, fixed in 4% paraformaldehyde, and subjected to micro-CT scanning using the Quantum GX2 imaging system. Data were analyzed using AccuCT™ software (PerkinElmer).

Histological Analysis

Following micro-CT scanning, knee joint tissues were decalcified in 0.5M ethylenediaminetetraacetic acid (EDTA, pH 8.0) for twentyone days, with the solution changed every two to three days. After decalcification, tissues were dehydrated through a graded ethanol series, cleared in xylene, and embedded in paraffin wax. Paraffin blocks were sectioned sagittally at nine μm thickness using a microtome. Safranin O/fast green staining was used to assess cartilage integrity. The severity of osteoarthritis was evaluated using the Osteoarthritis Research Society International (OARSI) scoring system and the Mankin score.

Statistical Analysis

Data were analyzed using R software (version 4.3.2) and presented as mean ± standard deviation (SD). Data was assessed for normality using the Shapiro–Wilk test, and a parametric method was applied. Comparisons between groups were made using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test for multiple comparisons. A p-value < 0.05 was considered statistically significant.

Results

Acupuncture Therapies Progressively Alleviate Pain Behaviors in MIA-Induced OA Rats

Paw withdrawal thresholds (PWT) were monitored over time to assess acupuncture’s impact on mechanical allodynia. Before MIA-injection, all groups exhibited similar baseline PWTs (15.2±0.9g), confirming no pre-existing differences. One-week post-MIA induction, all groups showed a rapid and sustained reduction in PWT, confirming pain hypersensitivity (Figure 1A) while the control group remained stable Acupuncture-treated rats showed progressive recovery. By Week 2, EA and LA groups showed modest improvements (p < 0.05; p<0.01 vs OA) while MA and BVA groups did not show any significant improvement. From Week 3 onward, LA and EA exhibited further gains (p < 0.01), with all three modalities demonstrating significant pain reversal by the final week (LA: p < 0.001; EA: p < 0.01; MA: p < 0.05). Weekly comparisons revealed that LA maintained a significant reduction from week two onwards, EA was improved on week two and four and MA started showing improvement from week four (Figure 1B).

Figure 1 Changes in paw withdrawal threshold over 4 weeks across different treatment groups in MIA-induced OA rats. (A) Changes in paw withdrawal threshold over time. (B) PWT values for groups from Week_1 to Week_4; n=8 per group. Data are presented as the mean ±standard deviation; p<0.05, p <0.05, p <0.01, p <0.001 compared with the OA group. Abbreviations: Con, control; OA, osteoarthritis; BVA, bee venom acupuncture; MA, manual acupuncture; EA, electro-acupuncture; LA, laser acupuncture.

Micro-CT Imaging Demonstrates Joint Preservation and Inhibition of Pathological Ossicle Formation

Micro-CT imaging revealed that untreated OA knees displayed classic signs of joint degeneration: subchondral erosion, trabecular irregularity, and surface damage (Figure 2). In contrast, the EA, LA, and BVA groups maintained better joint morphology, smoother bone contours, and preserved trabeculae.

Figure 2 Representative micro-CT 3D images. The control knee maintains trabecular subchondral plate integrity with a smooth contour; the OA knee exhibits clear bone erosion following MIA induction. n=8. Abbreviations: Con, control; OA, osteoarthritis; BVA, bee venom acupuncture; MA, manual acupuncture; EA, electro-acupuncture; LA, laser acupuncture.

Meniscal ossicles, indicative of OA progression, were significantly enlarged in OA rats (Figure 3). LA significantly reduced ossicle volume and area (p < 0.006 and p < 0.006 vs OA). EA, BVA and MA groups exhibited moderate effects.

Figure 3 Micro-CT analysis of the hind knee joint in MIA-induced OA rats; (A) Area, (B) Volume of the meniscal ossicles. The area and volume of meniscal ossicles, abnormal bone formations within the knee meniscus, across different groups compared to the OA group. Error bars represent standard deviation. p-values indicate significant differences compared to the OA group. n=8. Data are presented as the mean ±standard deviation; p <0.05, p <0.01, p <0.001. Abbreviations: Con, control; OA, osteoarthritis; BVA, bee venom acupuncture; MA, manual acupuncture; EA, electro-acupuncture; LA, laser acupuncture.

Laser and Electroacupuncture Preserve Cartilage Integrity

Safranin-O/Fast Green staining revealed severe cartilage erosion, proteoglycan loss, and chondrocyte disarray in OA rats (Figure 4). EA and LA groups retained matrix staining and structural integrity, similar to the control group. BVA and MA showed partial preservation.

Figure 4 Histological images and quantification of cartilage degradation using Safranin-O/Fast Green staining. (A) Sagittal sections of rat knee joints from each group were stained with Safranin-O/Fast Green. Red staining indicates proteoglycan-rich cartilage, while loss of staining denotes matrix degradation. The OA group showed severe cartilage erosion and proteoglycan loss, whereas the EA and LA groups retained staining patterns similar to the control. Upper row: 10× magnification (scale bars = 100 µm); Lower row: 20× magnification (scale bars = 50 µm). (B) Quantitative assessment of cartilage damage using a modified OARSI scoring system. Boxplots represent median and interquartile range, with individual data points shown. EA and LA groups exhibited significantly lower cartilage scores compared to the OA group (****p < 0.0001, ***p < 0.001, **p < 0.01; one-way ANOVA with Tukey’s post hoc test). Groups with different letters differ significantly, while groups that share a letter do not differ significantly (a-d) (p < 0.05). Abbreviations: Con, control; OA, osteoarthritis; BVA, bee venom acupuncture; MA, manual acupuncture; EA, electro-acupuncture; LA, laser acupuncture.

Quantitative scoring confirmed these findings (Figure 4). OA rats had significantly elevated cartilage damage scores (ANOVA, p < 2.2e−16). EA and LA had the lowest scores (p < 0.0001 vs OA), while BVA and MA showed intermediate reductions (p < 0.001, p < 0.01, respectively).

Discussion

Pain is often the earliest and most persistent symptom of OA, so this study focused on it. We set out to investigate whether EA, LA, and BVA could relieve pain and slow the progression of joint degeneration in MIA-induced OA rats. The results of our study not only confirm the potential of these acupuncture therapies and offer hope and optimism for the future of OA treatment.

Behaviorally, LA and EA improved pain thresholds by Week 2, and by Week 4, LA, EA, and MA significantly reversed mechanical hypersensitivity. Animals in the BVA group experienced some inflammation at the acupoint after treatment; this could account for the low PWT. These effects were verified by micro-CT findings, which showed preserved subchondral structure in EA and LA groups. Rarely assessed in preclinical acupuncture studies, ossicle formation was markedly inhibited by EA and LA, suggesting modulation of aberrant bone remodeling. Safranin-O staining further confirmed that EA and LA most effectively preserved cartilage integrity.

Previous studies have individually validated the efficacy of acupuncture treatments. For instance, Ma et al showed that early EA (at ST35/ST36) preserved cartilage and relieved pain, while delayed EA had reduced benefit.²⁷ Chen et al further revealed that EA acts via sympathetic β2-adrenergic signaling to suppress IL-6, reduce synovial inflammation, and ameliorate pain behaviors.²² Our results align with these findings, as EA improved PWT threshold by week 2 and preserved cartilage, as confirmed by the histology scores.

LA has shown promise in modulating inflammation and promoting cartilage repair.^21,28 Li et al demonstrated that 10.6 μm infrared LA reduced MMP-13 expression, improved weight-bearing, and preserved cartilage in MIA-OA rats, resulting in LA’s anti-inflammatory and chondroprotective potential.²⁹ In our study, invasive 830 nm LA produced similar benefits: pain thresholds improved significantly from Week 2, cartilage histology closely resembled that of the control group by Week 4 and reduced ossicle formation. These results suggest that LA, despite being a less invasive and more technologically modern modality, may offer outcomes comparable to EA in treating OA as the LA penetrates deeper into the skin.³⁰

 BVA has demonstrated significant analgesic and anti-inflammatory effects through pharmacological mechanisms. Chen and Larivière (2010) reviewed bee venom’s actions and noted its impact on opioid receptors and the suppression of proinflammatory cytokines such as TNF-α and IL-1β.³¹ Our study supports this mechanistic framework as BVA-treated rats exhibited early pain relief (Week 2) and moderate histological protection. However, its structural preservation was less pronounced than that observed with EA and LA.

A subset of rats in the BVA group developed localized swelling and reduced mobility following the initial bee venom injections. Such responses are consistent with documented side effects of bee venom therapy, which include local inflammation, edema, and, in some cases, systemic reactions. For instance, a systematic review highlighted that bee venom therapy can lead to adverse events ranging from mild local reactions to severe systemic responses, depending on the dosage and administration method. Additionally, studies have reported that bee venom injections can cause localized swelling and pain in animal models. These adverse reactions were not observed in the other treatment groups.^32,33

Our findings align with a network meta-analysis by Corbett et al (2013), which found acupuncture among the most effective non-pharmacological treatments for knee OA.¹⁶ While their analysis was limited to clinical studies and did not distinguish between acupuncture types, our data add nuance by suggesting that different modalities may yield comparable overall benefits through distinct mechanisms.

Few studies have directly compared EA and LA. Kim et al (2019) evaluated EA and LA in a collagenase-induced arthritis model and reported superior outcomes with LA.²⁰ However, they did not incorporate BVA or assess multiple modalities simultaneously within the same framework. By integrating all three therapies, our study addresses this critical gap and provides clinicians and researchers with comparative evidence to inform integrative treatment strategies.

Understanding how each acupuncture treatment performs could guide therapy selection. Since EA, LA, and BVA function through distinct mechanisms, such as electrical stimulation, PBM, and biochemical immune modulation, understanding their relative effects in one system may inform future combined or personalized protocols. We prioritized functional and structural outcomes over molecular testing to ensure that our findings were closely related to the clinical characteristics of OA.

Conclusion

Acupuncture reduced both pain behaviors and cartilage degeneration in MIA-induced KOA, but the benefit was contingent upon the specific modality employed. LA had the most substantial and persistent therapeutic results, as indicated by changes in pain thresholds, cartilage, and bone structure. Its effectiveness outperformed that of EA and MA. In contrast, BVA had little efficacy and caused acute adverse reactions. This study suggests that the therapeutic efficacy of acupuncture for KOA is determined by the modality used, with laser-based approaches outperforming other methods. These findings emphasize the potential for adapting acupuncture modalities to disease pathophysiology and the importance of integrative, comparative research in advancing complementary OA therapies.

Abbreviations

BVA, bee venom acupuncture; CT, computed tomography; EA, electroacupuncture; EDTA, ethylenediaminetetraacetic acid; GB34, gall bladder 34 acupoint (yanglingquan); LA, laser acupuncture, MA, manual acupuncture; MIA, monosodium iodoacetate; OA, osteoarthritis; PWT, paw withdrawal threshold; PBM, photobiomodulation.

Data Sharing Statement

The data generated for the present study are available from the corresponding author, Gihyun Lee: [email protected], and Jae-Hong Kim: [email protected], upon reasonable request.

Ethical Statement

This study was approved by the animal care and use committee of Dongshin University (DSU-2024-07-04).

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Funding

This research was supported by a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: RS-2023-KH139215).

Disclosure

The authors declare that they have no conflicts of interest in this work.

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Introduction

Inflammatory Bowel Disease (IBD) is a chronic, relapsing condition that primarily includes Crohn’s disease (CD) and ulcerative colitis (UC).¹ IBD most commonly affects young adults, with peak incidence between the ages of 15 and 30 years, and a smaller peak occurring in later adulthood.^2,3 These diseases are characterized by inflammation of the gastrointestinal tract, leading to significant morbidity and disability.⁴ Symptoms such as abdominal pain, diarrhea, and weight loss can severely impact quality of life and result in substantial healthcare costs. Globally, the economic burden of IBD is substantial, with significant implications for healthcare systems worldwide.^5–9 In the United States alone, IBD affects over 3 million individuals and is a leading cause of death and disability among digestive diseases.¹⁰ Despite a decrease in prevalence from 1990 to 2019, the mortality rate for IBD in the US increased by 172%, and Disability Adjusted Life Years (DALYs) rose by 16%, indicating a growing burden on the healthcare system.¹⁰ The economic impact of IBD is multifaceted, involving direct costs such as hospitalizations, medications, and outpatient care, as well as indirect costs related to lost productivity and long-term disability.

Compared with traditional therapies such as corticosteroids, aminosalicylates, and immunomodulators, which mainly provide nonspecific immunosuppression or symptomatic relief, biologic therapies offer targeted modulation of key inflammatory pathways.⁴ With the advent of biologic therapies, there has been a substantial improvement in the management of IBD.^11,12 Biologics, such as anti-tumor necrosis factor (TNF) agents, have revolutionized the treatment landscape by providing effective control of inflammation and reducing the need for surgery. These therapies have enabled many patients to achieve and maintain remission, significantly improving their quality of life and long-term outcomes.

However, treating IBD in women of reproductive age presents unique challenges. The safety of biologic therapies during pregnancy and breastfeeding is a critical concern that requires careful consideration and management.^13–15 Evidence indicates that maintaining remission is crucial, as uncontrolled IBD itself increases risks of preterm birth and low birth weight. Anti-TNF agents and newer IL-23 inhibitors are generally considered safe, while discontinuation during pregnancy raises relapse risk.¹³ Surgical history also matters, since procedures such as ileal pouch–anal anastomosis (IPAA) can reduce fertility.¹⁵ In addition, women with IBD have higher rates of cesarean section and adverse maternal outcomes compared with women without IBD.¹⁵

Moreover, IBD can profoundly impact the psychological well-being of women in this age group, who often face additional stress due to their roles in family and work.¹⁶ The burden of IBD may significantly affect their social roles and overall quality of life.^17,18 Therefore, understanding the epidemiology of IBD specifically in women of reproductive age is essential for developing targeted interventions and support mechanisms. In particular, by disaggregating the reproductive-age population into seven 5-year age bands and applying the Nordpred model for age-specific projections, this study offers novel insights beyond prior GBD-based analyses.

Methods

Data Source

This study utilizes data from the 2021 Global Burden of Disease (GBD) study, which provides comprehensive estimates on the incidence, prevalence, years lived with disability (YLDs), DALYs, and healthy life expectancy (HALE) for 371 diseases and injuries across 204 countries and territories, encompassing 811 subnational regions.^19,20 Specifically, our research examines the burden of IBD among women of reproductive age from 1992 to 2021. The age groups included in this study are: 15–19, 20–24, 25–29, 30–34, 35–39, 40–44, and 45–49 years. Data collected includes the number of cases, incidence rates, mortality rates, and DALY rates. This research adheres to the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines for reporting observational epidemiological studies.

Socio-Demographic Index (SDI)

Developed by GBD researchers, the SDI is a composite indicator of development status that is strongly correlated with health outcomes. The SDI is the geometric mean of three key indices, each ranging from 0 to 1: the total fertility rate under the age of 25 (TFU25), the mean education level for individuals aged 15 and older (EDU15+), and the lag-distributed income (LDI) per capita. A location with an SDI of 0 represents a theoretical minimum level of development relevant to health, while an SDI of 1 represents a theoretical maximum level. In this study, we used SDI to stratify countries and regions into five categories (low, low-middle, middle, high-middle, and high) to analyze the burden of IBD among women of reproductive age. This stratification helps to understand how socio-demographic factors influence the incidence, prevalence, mortality, and DALYs associated with IBD in different development settings.

Statistical Analysis

We conducted a comprehensive statistical analysis to assess the burden of IBD among women of reproductive age from 1992 to 2021. Incidence rates, mortality rates, and DALYs rates were calculated per 100,000 population for each year and age group (15–19, 20–24, 25–29, 30–34, 35–39, 40–44, and 45–49 years). The 95% uncertainty intervals (UIs) for these estimates were derived from 1000 bootstrap replications to account for variability and ensure robustness.¹⁹

To evaluate trends over time, we computed the estimated annual percentage change (EAPC) for incidence rates, mortality rates, and DALYs rates using joinpoint regression analysis. We investigated the relationship between socio-demographic factors and the burden of IBD by stratifying data according to the SDI. To explore non-linear associations between SDI and IBD outcomes, we used loess smoothing. Additionally, Spearman’s rank correlation was applied to examine linear trends and the impact of different SDI levels on the burden of IBD.²¹ In our predictive analysis, we employed the Nordpred model, which utilizes age-period-cohort analysis to forecast future disease trends. This method provides a structured framework for estimating future health scenarios based on historical data and demographic dynamics.²² Our analysis was conducted on a global scale, with a specific focus on forecasting IBD trends across different age groups of women of reproductive age. This approach allows us to capture both the overall global burden and the specific impacts within this critical demographic. All analyses were done in R 4.3.3 with packages: ggplot2, sf, segmented, broom, dplyr, tidyr, INLA, BAPC.

Results

Inflammatory Bowel Disease in Women of Reproductive: Global Trends

Incidence

In 2021, the global incidence of IBD among women of reproductive age was 98,974.56 cases (95% UI: 80,567.63–124,088.76). This represents a 55.04% increase from the 63,839.03 cases (95% UI: 52,841.67–78,582.08) reported in 1992. The incidence rate increased from 4.61 per 100,000 population (95% UI: 3.82–5.68) in 1992 to 5.08 per 100,000 population (95% UI: 4.13–6.37) in 2021, with an EAPC of 0.43 (95% CI: 0.28–0.58) (Table 1). In 2021, the highest incidence rate was observed in the age group of 45–49 years, with a rate of 8.11 per 100,000 population, whereas the lowest incidence rate was found in the 15–19 years age group, with a rate of 1.40 per 100,000 population (Figure 1). Between 1992 and 2021, the age group 40–44 years experienced the most significant change in incidence rates, with an increase of 0.53 per 100,000 population. Conversely, the 25–29 years age group had the smallest change, with an increase of only 0.05 per 100,000 population (Figure 2).

Table 1 Incidence of Inflammatory Bowel Disease Among Women of Reproductive Age at the Global and Regional Levels Between 1992 and 2021

Figure 1 Incidence, Death, and DALYs Numbers and Rates of Inflammatory Bowel Disease Among Women of Reproductive Age in 2021. (A) Number and rate of incidence; (B) Number and rate of death; (C) Number and rate of DALYs.

Figure 2 Trends in Inflammatory Bowel Disease Incidence, Death, and DALYs rates among women of reproductive age from 1992 to 2021. (A) Rate of incidence; (B) Rate of death; (C) Rate of DALYs.

Mortality

In 2021, the global number of deaths due to IBD among women of reproductive age was 2,586.76 (95% UI: 1,900.32–3,125.77). This represents a 40.83% increase from the 1,836.75 deaths (95% UI: 1,195.53–2,384.65) reported in 1992. The death rate remained relatively stable, with 0.13 deaths per 100,000 population (95% UI: 0.09–0.17) in 1992 and 0.13 deaths per 100,000 population (95% UI: 0.10–0.16) in 2021, with an EAPC of −0.07 (95% CI: −0.12 to −0.01) (Supplement Table 1). In 2021, the highest death rate was observed in the age group of 40–44 years, with a rate of 0.22 per 100,000 population, whereas the lowest death rate was found in the 15–19 years age group, with a rate of 0.03 per 100,000 population (Figure 1). Between 1992 and 2021, the age group 45–49 years experienced the most significant decrease in death rates, with a reduction of 0.08 per 100,000 population. Conversely, the 30–34 years age group had the smallest decrease, with a reduction of only 0.002 per 100,000 population (Figure 2).

DALYs

In 2021, the global number of DALYs due to IBD among women of reproductive age was 281,580.36 (95% UI: 223,989.19–349,965.81). This represents a 38.53% increase from the 203,259.52 DALYs (95% UI: 154,006.13–257,799.02) reported in 1992. The DALYs rate slightly decreased from 14.68 per 100,000 population (95% UI: 11.12–18.62) in 1992 to 14.45 per 100,000 population (95% UI: 11.49–17.96) in 2021 (Supplement Table 2). In 2021, the highest DALYs rate was observed in the age group of 40–44 years, with a rate of 23.38 per 100,000 population, whereas the lowest DALYs rate was found in the 15–19 years age group, with a rate of 3.19 per 100,000 population (Figure 1). Between 1992 and 2021, the age group 45–49 years experienced the most significant decrease in DALYs rates, with a reduction of 5.22 per 100,000 population. Conversely, the 25–29 years age group had the smallest change, with a decrease of only 0.001 per 100,000 population (Figure 2).

Inflammatory Bowel Disease in Women of Reproductive: Socio-Demographic Index Levels

Incidence

In 2021, the number of prevalent cases of IBD among women of reproductive age was highest in the High SDI region, with a total of 38,912.17 cases (95% UI: 32,056.09–47,182.84). The High SDI region also had the highest prevalence rate, at 16.00 per 100,000 population (95% UI: 13.18–19.40). The most significant increase in the number of cases from 1992 to 2021 was observed in the Middle SDI region, with an absolute increase of 9,764.68 cases. The Middle SDI region also exhibited the highest EAPC, with a value of 2.07 (95% CI: 1.81–2.33) (Table 1).

Mortality

From 1992 to 2021, the regions that experienced a decline in mortality rates for IBD among women of reproductive age, as indicated by the EAPC, included the High-middle SDI region with an EAPC of −1.64 (95% CI: −1.83 to −1.45), the Low-middle SDI region with an EAPC of −0.35 (95% CI: −0.42 to −0.28), and the Middle SDI region with an EAPC of −0.47 (95% CI: −0.60 to −0.34). In contrast, the High SDI region saw an increase in mortality rates, with an EAPC of 0.41 (95% CI: 0.06 to 0.75), as did the Low SDI region, with an EAPC of 0.13 (95% CI: 0.05 to 0.21). In 2021, the highest mortality rate was observed in the Low SDI region, at 0.27 per 100,000 population (95% UI: 0.16–0.36), whereas the lowest mortality rate was found in the High-middle SDI region, at 0.07 per 100,000 population (95% UI: 0.06–0.09) (Supplement Table 1).

DALYs

From 1992 to 2021, the EAPC analysis revealed that the Middle SDI region experienced the most significant increase in DALYs, with an EAPC of 0.39 (95% CI: 0.31–0.47). Conversely, the High-middle SDI region saw the most substantial decrease in EAPC, registering −0.39 (95% CI: −0.47 to −0.32). In 2021, the High SDI region exhibited the highest DALYs value for women of reproductive age (15–49 years) with IBD, reaching 75,867.33 (95% UI: 53,581.20–102,607.88). In contrast, the High-middle SDI region had the lowest DALYs value in 2021, recorded at 31,075.70 (95% UI: 23,755.44–40,083.38) (Supplement Table 2).

Inflammatory Bowel Disease in Women of Reproductive: Geographic Regional Trends

Incidence

In 2021, the highest number of incident cases of IBD among women of reproductive age was reported in High-income North America, with 19,864.53 cases (95% UI: 16,479.19–24,224.81). In contrast, Oceania had the fewest cases, with 30 cases (95% UI: 24–39). The region with the highest EAPC in incidence rates was East Asia, showing substantial growth over the period, with an EAPC of 3.40 (95% CI: 2.67–4.13). Oceania exhibited the lowest EAPC at 0.59 (95% CI: 0.55–0.62) (Table 1).

In 2021, the highest incidence rate was observed in High-income North America, with a rate of 23.64 per 100,000 population (95% UI: 19.61–28.83), while Oceania had the lowest incidence rate at 0.87 per 100,000 population (95% UI: 0.68–1.13). Among the 21 GBD regions, 8 regions had incidence rates above the global mean of 5.08 per 100,000 population (95% UI: 4.13–6.37), such as High-income North America, Southern Latin America, and Western Europe. In contrast, 13 regions, including Southeast Asia and East Asia, had incidence rates below the global mean (Table 1). Overall, a positive correlation is evident between SDI levels and IBD incidence rates, indicating that more developed regions experience higher rates of this disease (Figure 3).

Figure 3 Association SDI and Rates of Incidence, Death, and DALYs for Inflammatory Bowel Disease Among Women of Reproductive Age Across 21 GBD Regions from 1992 to 2021. (A) Incidence rate. (B) Death rate. (C) DALYs rate.

Mortality

In 2021, the highest number of deaths due to IBD among women of reproductive age was reported in South Asia, with 544.17 deaths (95% UI: 348.04–934.81). Oceania reported the fewest deaths, with 0.59 cases (95% UI: 0.31–0.94). The highest EAPC in mortality rates was in Australasia, with an EAPC of 2.69 (95% CI: 1.67–3.73), while High-income Asia Pacific had the lowest EAPC at −3.48 (95% CI: −3.78 to −3.17) (Supplement Table 1).

The highest mortality rate in 2021 was in Central Sub-Saharan Africa, at 0.09 per 100,000 population (95% UI: 0.05–0.14), and the lowest was in High-income North America, at 0.20 per 100,000 population (95% CI: 0.19–0.21). Among the 21 GBD regions, 7 regions had mortality rates above the global mean of 0.13 per 100,000 population (95% UI: 0.10–0.16), including Western Sub-Saharan Africa and Eastern Europe, while 14 regions, such as Southeast Asia and Australasia, had rates below the global mean (Supplement Table 1). Overall, the data reveal a strong inverse relationship between SDI levels and IBD mortality rates, highlighting that less developed regions are more heavily burdened by mortality due to IBD (Figure 3).

DALYs

In 2021, the highest number of DALYs due to IBD among women of reproductive age was reported in South Asia, with 67,327.10 DALYs (95% UI: 48,638.08–92,097.98). Oceania reported the fewest DALYs, with 68.95 DALYs (95% UI: 48.66–94.67) (Supplement Table 2).

The highest EAPC in DALY rates was in Central Latin America, with an EAPC of 1.15 (95% CI: 0.98–1.32), while Andean Latin America had the lowest EAPC at −1.57 (95% CI: −1.87 to −1.26). Among the 21 GBD regions, 9 regions had DALY rates above the global mean of 14.45 per 100,000 population, including Western Sub-Saharan Africa and Australasia, while 12 regions, such as Southeast Asia and East Asia, had rates below the global mean (Supplement Table 2). The graph depicts a U-shaped curve, indicating that both low and high SDI regions have higher DALY rates due to IBD, while middle SDI regions have lower rates (Figure 3).

Inflammatory Bowel Disease in Women of Reproductive: National Trends

Incidence

In 2021, the highest number of incident cases of IBD among women of reproductive age was reported in India, with 22,540.84 cases (95% UI: 18,131.75–28,645.95). The country with the highest EAPC in incidence rates was China, with an EAPC of 3.45 (95% CI: 2.71–4.19). On the other hand, Finland had the lowest EAPC at −1.85 (95% CI: −2.28 to −1.42) (Supplement Table 3).

The incidence rates of IBD among women of reproductive age in 2021 demonstrate a positive correlation with the SDI across 204 countries. High SDI countries, such as the Netherlands (32.38 per 100,000 population), Germany (26.99 per 100,000 population), and Norway (25.76 per 100,000 population), show relatively high incidence rates. In contrast, low SDI countries, such as the Lao People’s Democratic Republic (0.82 per 100,000 population) and Cambodia (0.83 per 100,000 population), exhibit significantly lower incidence rates. Middle SDI countries, including Kazakhstan (6.63 per 100,000 population) and the Kyrgyz Republic (5.28 per 100,000 population), present moderate incidence rates (Supplement Table 3). This pattern indicates that higher levels of socioeconomic development are associated with increased incidence rates of IBD (Figure 4).

Figure 4 Continued.

Figure 4 Relationship Between SDI and Rates of Incidence, Death, and DALYs for Inflammatory Bowel Disease Among Women of Reproductive Age in 204 Countries and Territories in 2021. (A) Incidence rates. (B) Death rates. (C) DALYs rates.

Mortality

In 2021, the highest number of deaths due to IBD among women of reproductive age was reported in India, with 357.21 deaths (95% UI: 203.56–652.99). The country with the highest EAPC in mortality rates was American Samoa, with an EAPC of 4.02 (95% CI: 2.84–5.22). Conversely, Singapore had the lowest EAPC at −8.05 (95% CI: −8.50 to −7.60) (Supplement Table 4).

The mortality rates due to IBD across countries in 2021 illustrate an inverse relationship with SDI levels. High SDI countries, such as Canada (0.13 per 100,000 population) and Australia (0.10 per 100,000 population), report relatively low mortality rates. In contrast, low SDI countries, such as Nigeria (0.56 per 100,000 population) and Ghana (0.71 per 100,000 population), experience significantly higher mortality rates. Middle SDI countries, including Kazakhstan (0.27 per 100,000 population) and Brazil (0.23 per 100,000 population), display moderate mortality rates (Supplement Table 4). This pattern highlights how socioeconomic development influences IBD mortality rates, with more developed regions achieving lower rates (Figure 4).

DALYs

In 2021, the highest number of DALYs due to IBD among women of reproductive age was reported in India, with 48,130.70 DALYs (95% UI: 34,218.65–67,011.34). The highest DALY rate was observed in Guinea-Bissau, with a rate of 87.33 per 100,000 population (95% UI: 47.06–141.34), while the lowest DALY rate was in the Solomon Islands, at 1.72 per 100,000 population (95% UI: 1.18–2.37). The country with the highest EAPC in DALY rates was Libya, with an EAPC of 2.67 (95% CI: 2.42–2.93). Conversely, Estonia had the lowest EAPC at −2.64 (95% CI: −3.66 to −1.60) (Supplement Table 5).

The DALY rates due to IBD across countries in 2021 demonstrate a U-shaped pattern when examined in relation to SDI levels (Figure 4). High SDI countries, such as Canada (76.20 per 100,000 population) and the United States (38.67 per 100,000 population), exhibit relatively elevated DALY rates. Similarly, low SDI countries, such as Nigeria (34.37 per 100,000 population) and Ghana (43.54 per 100,000 population), also report elevated DALY rates. In contrast, middle SDI countries, including Kazakhstan (24.27 per 100,000 population), Brazil (16.14 per 100,000 population), and Turkey (12.01 per 100,000 population), display moderate DALY rates (Supplement Table 5).

Inflammatory Bowel Disease in Women of Reproductive Age: Projected Global Trends (2021–2030)

From 2021 to 2030, the global burden of IBD among women of reproductive age is projected to decline gradually. In 2021, the number of IBD cases was estimated at 98,975, with an incidence rate of 5.08 per 100,000. By 2030, this number is expected to decrease to 94,773, with a corresponding incidence rate of 4.51 per 100,000. Despite this decrease, the number of deaths attributable to IBD is anticipated to rise slightly, from 2587 in 2021 to 2781 in 2030, while the mortality rate will remain stable at around 0.132 per 100,000. The DALYs associated with IBD are expected to increase slightly from 281,580 years in 2021 to 284,508 years in 2030, with the DALY rate changing from 14.45 per 100,000 in 2021 to 13.55 per 100,000 in 2030 (Supplement Table 6).

Age-specific trends reveal that the incidence of IBD increases progressively with age, peaking in the 45 to 49 age group by 2030, where the number of cases is projected to reach 21,213, with an incidence rate of 7.79 per 100,000. The incidence remains high in the 40 to 44 age group as well, with 20,736 cases and an incidence rate of 6.96 per 100,000. Correspondingly, the death rate is expected to escalate from 0.03 per 100,000 in the 15 to 19 age group to 0.21 per 100,000 in the 45 to 49 age group, with the 40 to 44 age group seeing a similar rate of 0.22 per 100,000. The DALYs rate will similarly rise with age, starting at 2.95 per 100,000 in the youngest group (15–19 years) and peaking at 21.42 per 100,000 in the 45 to 49 age group, closely followed by 21.18 per 100,000 in the 40 to 44 age group. These trends underscore the increasing burden of IBD with advancing age, particularly in the older reproductive age groups (Figure 5).

Figure 5 Global trends and projections of incidence, Death, and DALYs of inflammatory bowel disease among women of reproductive age (1992–2030). (A) Number and rate of incidence; (B) Number and rate of death; (C) Number and rate of DALYs.

Discussion

The burden of IBD has been increasing steadily, presenting a significant public health challenge,^5,7 particularly among women of reproductive age.²³ This demographic is unique due to the intersection of their reproductive health and the management complexities of IBD.²⁴ Our study conducted a comprehensive analysis of the global, regional, and national burden of IBD from 1992 to 2021, focusing on incidence, mortality, and DALYs. We further explored how these indicators varied across different regions and countries according to their SDI levels. This study provides crucial epidemiological evidence necessary for developing targeted public health strategies and interventions aimed at mitigating the impact of IBD on women of reproductive age, ultimately improving their quality of life and health outcomes.

Globally, the incidence, mortality, and DALYs associated with IBD among women of reproductive age have shown significant trends from 1992 to 2021. The incidence of IBD increased by 55.04%, from 63,839.03 cases in 1992 to 98,974.56 cases in 2021, with the incidence rate rising from 4.61 to 5.08 per 100,000 population. The highest incidence rate in 2021 was observed in women aged 45–49 years, whereas the lowest was in those aged 15–19 years. This suggests that older women within the reproductive age spectrum are more frequently diagnosed with IBD. As women age, the decline in immune regulation and significant alterations in gut microbiota may synergistically contribute to the increased risk and higher incidence of IBD.^25,26

Despite the increase in incidence, the overall mortality rate remained relatively stable, staying at around 0.13 deaths per 100,000 population. The highest mortality rate was observed in the 40–44 years age group, reaching 0.22 deaths per 100,000 population. However, DALYs increased by 38.53%, indicating a growing burden of disability. The age group of 45–49 years experienced the most significant increase in DALYs, highlighting the prolonged impact of IBD on older women within the reproductive age. This could be due to the chronic nature of the disease and its complications, including frequent relapses, long-term medication requirements, and surgeries, all of which impact quality of life.^27–29 Public health interventions must address these growing burdens to improve outcomes and quality of life for this vulnerable population.

The burden of IBD among women of reproductive age varies significantly across different socioeconomic contexts, as illustrated by the SDI. In regions with higher SDI, such as North America, Western Europe, and Australia, the incidence of IBD is markedly elevated. For instance, in economically developed regions like North America, IBD stands as a leading cause of disability and mortality among digestive diseases. This substantial disease burden necessitates robust healthcare interventions. Contributing to this higher incidence is the heightened awareness and availability of advanced diagnostic tools in these regions, including endoscopy, fecal calprotectin, and gastrointestinal ultrasound, which enhance IBD detection.^30,31 These diagnostic tools, combined with a high index of suspicion in primary care settings, facilitate earlier and more frequent diagnoses, potentially inflating incidence rates compared to regions with less developed healthcare infrastructures. Furthermore, these regions benefit from early detection, comprehensive management protocols, and greater access to advanced therapies, which together contribute to lower mortality rates, despite the higher prevalence of the disease.

Beyond diagnostic advances, therapeutic innovations—particularly the introduction of biologics since the late 1990s—have substantially improved the prognosis of IBD.³² Infliximab was first approved for Crohn’s disease in 1998 and subsequently for ulcerative colitis, with newer biologics such as IL-12/23 and IL-23 inhibitors further broadening treatment options.³³ Evidence from clinical trials and population-based studies indicates that biologics reduce hospitalization, surgery, and mortality, while improving quality-adjusted life years (QALYs).³⁴ These advances may help explain the stabilization or decline in mortality despite rising incidence, particularly in high-SDI regions where access to biologics is greater. Although GBD data cannot directly capture treatment effects, acknowledging these developments provides important context for interpreting observed trends and emphasizes the need for equitable access in reproductive-age women.

In contrast, lower SDI regions, such as Western Sub-Saharan Africa, exhibit lower incidence rates but face disproportionately higher mortality and DALY rates. This disparity is largely driven by inadequate healthcare infrastructure, limited access to advanced therapies, and delays in diagnosis, which exacerbate the disease burden.^34–36 Middle SDI regions, including Central Asia and Latin America, are currently navigating a transitional phase characterized by improvements in healthcare systems that have begun to enhance disease identification and management. Despite these advancements, these regions continue to face significant challenges in managing the chronic and debilitating nature of IBD, as reflected by the substantial increase in DALYs. This growing burden is compounded by the rapid urbanization and westernization in these areas, which contribute to a rising incidence of IBD.⁸ Addressing these disparities is essential for improving health outcomes and the quality of life for women of reproductive age affected by IBD, particularly as these regions confront the dual challenges of increasing prevalence and an aging population.

Finally, the projected trends in the global burden of IBD among women of reproductive age from 2021 to 2030 indicate important shifts across different age groups. While the overall incidence rate is projected to decrease slightly, the total number of cases remains substantial, particularly in the older segment of this age range. The mortality rate is expected to remain stable, yet an increase in the number of deaths is anticipated, reflecting population growth within this demographic. Additionally, a slight increase in DALYs is expected, especially among those aged 40–49, underscoring the ongoing challenge posed by the chronic nature of IBD. These projections highlight the necessity for targeted public health interventions, focusing on early diagnosis, effective disease management, and comprehensive support, to alleviate the long-term impact of IBD on this population.

Limitations

This study has several limitations. First, the reliance on GBD 2021 data, which incorporates modeled estimates, may introduce inaccuracies, particularly in low-income regions where data quality is less reliable. This could lead to an underestimation of the true burden of IBD in these areas. Additionally, the projections for IBD burden are based on historical trends and do not consider potential future interventions or policy changes that could alter these trends. Furthermore, the study was unable to analyze specific risk factors for IBD due to the limitations of the database used, which could have provided more insights for targeted interventions. Finally, ecological and environmental factors such as latitude, UV exposure, and vitamin D—known to be associated with IBD epidemiology—could not be assessed, as these indices are not included in the GBD 2021 dataset.

Conclusion

In conclusion, the global burden of IBD among women of reproductive age has significantly increased from 1992 to 2021, with the total number of cases rising by 55% and incidence rates showing a steady upward trend. This growth highlights the ongoing challenges posed by IBD on a global scale. Substantial variations are evident across different SDI regions. High SDI regions, while experiencing higher incidence rates and DALYs, have relatively lower mortality rates. In contrast, low SDI regions face a disproportionate burden, with higher mortality and DALYs despite lower incidence rates. Looking forward, projections to 2030 suggest a slight decrease in global incidence rates; however, the absolute number of cases and related deaths is expected to continue increasing, particularly among older women within the reproductive age range. These findings emphasize the urgent need for targeted public health interventions that focus on early diagnosis, effective management strategies, and comprehensive support systems, particularly in resource-limited settings.

Abbreviation

IBD, Inflammatory Bowel Disease; GBD, Global Burden of Disease; DALYs, Disability-Adjusted Life Years; EAPC, Estimated Annual Percentage Change; SDI, Socio-Demographic Index; CD, Crohn’s disease; UC, Ulcerative Colitis; TNF, Tumor Necrosis Factor; YLDs, Years Lived with Disability; HALE, Healthy Life Expectancy.

Data Sharing Statement

The data can be obtained from a public, open-access database. Information regarding data access policies and procedures can be found at https://ghdx.healthdata.org/gbd-2021.

Ethics Approval and Consent to Participate

This study used only de-identified, publicly available data from the Global Burden of Disease (GBD) 2021 database. The Ethics Committee of Xiangyang No.1 People’s Hospital determined that formal approval was not required.

Acknowledgments

We would like to express our heartfelt appreciation to the Global Burden of Disease Collaborative Network and the Institute for Health Metrics and Evaluation (IHME) for their invaluable support. We sincerely thank the editor and reviewers for their valuable feedback, which has greatly improved the quality of our manuscript.

Author Contributions

All authors made a significant contribution to the work reported, whether in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising, or critically reviewing the article; gave final approval of the version to be published; agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Funding

No external funding was received for the study.

Disclosure

The authors declare that they have no competing interests in this work.

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The Dutch government said on Sunday that it had taken the “highly exceptional” decision to intervene at Chinese-owned chipmaker Nexperia over a potential “risk to Dutch and European economic security.”

The Netherlands-based firm’s owner Wingtech said on Monday that it will take actions to protect its rights and will seek government support.

The development threatens to raise tensions between the European Union and China, which have increased in recent months over trade and Beijing’s relationship with Russia.

Nexperia was forced to sell its silicon chip plant in Newport, Wales after MPs and ministers expressed national security concerns. It currently owns a UK facility in Stockport.

The Dutch government said its economic affairs ministry had invoked its Goods Availability Act over “acute signals of serious governance shortcomings” within Nexperia.

The law is designed to allow the Hague to intervene in companies under exceptional circumstances. These include threats to the country’s economic security and to ensure the supply of critical goods.

The intervention is meant to prevent a potential situation in which Nexperia’s chips would become unavailable in an emergency, said the Dutch government.

It added that Nexperia’s operations posed a “threat to the continuity and safeguarding on Dutch and European soil of crucial technological knowledge and capabilities.”

The company’s production can continue as normal, it added.

Nexperia makes semiconductors used in cars and consumer electronics.

The government statement did not detail why it thought the firm’s operations were risky. The BBC has contacted Dutch authorities for clarification.

Shanghai-listed shares in Nexperia’s parent company Wingtech fell by 10% on Monday morning.

Wingtech is among the firms the US has placed on its so-called “entity list”. Under the regulations, US companies are barred from exporting American-made goods to businesses on the list unless they have special approval.

In September, the US commerce department further tightened its restrictions, adding to the entity list any company that is majority-owned by a Chinese firm.

Introduction

Hepatocellular carcinoma (HCC) ranked as the third leading cause of cancer mortality globally in 2020, accounting for 75%-85% of primary liver cancers.¹ Treatment modalities for HCC encompass surgical resection, chemotherapy, transcatheter arterial chemoembolization (TACE), and systemic therapy. However, most HCC cases are diagnosed at advanced stages, missing the optimal window for surgical intervention. Even post-resection, HCC exhibits alarming 5-year recurrence rates ranging between 60%-70%.² Metastasis and relapse are primary factors that significantly impact patients’ long-term survival, posing a critical challenge in the overall management of HCC.³ In recent years, traditional Chinese medicine (TCM) has gained attention as a complementary approach in HCC treatment due to its multi-targeted mechanisms, including modulation of tumor growth, metastasis, and immune responses, which may improve therapeutic efficacy and reduce adverse effects.⁴ Therefore, a comprehensive understanding of the molecular mechanisms orchestrating the metastatic cascade is crucial for advancing HCC treatment, potentially revealing new therapeutic targets and integrative strategies.

Tumor metastasis remains the leading cause of cancer-related mortality worldwide.⁵ During this complex process, tumor cells must detach from the primary lesion, degrade the extracellular matrix (ECM), intravasate into the bloodstream, survive circulatory stress, extravasate, and ultimately colonize distant organs.⁶ Among the many factors influencing this metastatic cascade, anoikis resistance and hypoxia are two critical stress responses that significantly contribute to tumor progression.^7,8 Anoikis, or anchorage-dependent programmed cell death, is typically induced when epithelial cells lose contact with the ECM.⁷ However, epithelial-derived tumor cells frequently acquire anoikis resistance during malignant transformation, particularly in metastatic settings, which enables them to survive in suspension, traverse the vasculature, and establish metastases.⁹ HCC, which arises from epithelial hepatocytes and exhibits strong vascularity, often displays features of anoikis resistance, facilitating hematogenous dissemination.¹⁰ In parallel, hypoxia is a hallmark of the solid tumor microenvironment (TME) and plays a pivotal role in promoting tumor aggressiveness. HCC frequently experiences intratumoral hypoxia due to rapid proliferation and abnormal vasculature.¹¹ Hypoxia not only enhances invasion and migration but also sustains cancer stemness through the activation of oncogenic pathways such as Wnt/β-catenin.¹² Furthermore, hypoxia profoundly reshapes the tumor immune microenvironment (TIME) by modulating immune cell infiltration, inducing immunosuppressive phenotypes, and promoting immune evasion, which collectively facilitate tumor progression and metastasis.¹³ Although both hypoxia and anoikis resistance are critical in HCC progression, they are often studied separately, with limited focus on their combined effects.

Long non-coding RNA (lncRNA), a class of transcripts longer than 200 nucleotides, have emerged as important regulators of tumor biology, including proliferation, metastasis, recurrence, prognosis, and therapeutic response.^14–17 LncRNAs can be functionally categorized into immune-related, hypoxia-responsive, EMT-related, and anoikis-associated types.^18–21 For instance, hypoxia-responsive lncRNAs such as LINC00839 are transcriptionally activated under oxygen-deprived conditions and modulate tumor proliferation and immune evasion.¹⁸ Likewise, anoikis-related lncRNAs such as AL031985.3 and AC026412.3 promote anchorage-independent survival and enhance metastatic potential.²¹ Although both hypoxia- and anoikis-related lncRNAs have been individually studied in HCC, integrated analyses remain scarce. To the best of our knowledge, no prior studies have systematically combined hypoxia- and anoikis-related lncRNA signatures to define molecular subtypes or predict immune landscape and prognosis in HCC. Given the converging effects of these stress responses on tumor stemness, immune suppression, and metastasis, their integration may yield a more comprehensive understanding of tumor biology and guide treatment stratification.

In this investigation, hypoxia- and anoikis-related lncRNAs were identified, and gene expression datasets and clinical data of liver cancer patients were retrieved from TCGA GDC API and GSE43619 databases. The study aimed to explore the interplay between hypoxia- and anoikis-related lncRNAs and the prognosis of HCC patients. By utilizing hypoxia- and anoikis-related lncRNAs, HCC was stratified into two molecular subtypes, with comparative evaluations of immunophenotypic characteristics across these subsets. Furthermore, a prognostic model centered around hypoxia- and anoikis-related lncRNAs was developed to decipher their associations with HCC prognosis and tumor immunophenotype (Figure 1). This research effort contributes to understanding the implications of hypoxia- and anoikis-related lncRNAs in HCC, unveiling new avenues for metastasis biomarkers and clinical interventions.

Figure 1 Research process. (A) Identification of Hypoxia- and anoikis-related lncRNA genes. (B) Construction and validation of gene prognostic model. (C) Experimental verification.

Materials and Methods

Data Sources

RNA-seq data from TCGA GDC API (https://gdc.cancer.gov/developers/gdc-application-programming-interface-api) were utilized to download expression data and clinical follow-up information of LIHC samples. The RNA-Seq data from TCGA-LIHC removed samples without survival time and status, converted Ensembl to Gene symbol, transformed the expression matrix into TPM format, and performed log2 conversion. The TCGA-LIHC cohort was used for the construction and internal validation of the risk model. Additionally, gene expression data and clinical information from the GSE188608 and GSE103581 cohorts were downloaded from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/). These datasets were specifically used to identify hypoxia- and anoikis-related lncRNAs.

The GSE43619 dataset was also obtained from GEO and used for external validation of the constructed prognostic model. Platform-specific annotation files were used to map probe IDs to gene symbols, and the average expression value was taken when multiple probes corresponded to a single gene. Subsequently, 365 HCC samples and 50 adjacent control samples were acquired. For the GSE43619 data, the annotation information of the corresponding chip platform was downloaded, and probes were mapped to genes where the mean value was considered as the gene expression. This research utilized secondary datasets that had been fully de-identified and contained no personally identifiable information; therefore, ethical approval was not required. As the data originated from publicly accessible genomic databases and the study did not involve any direct contact with human subjects or implementation of invasive procedures, informed consent was waived. In accordance with relevant ethical guidelines on the use of public data, this study meets the criteria for exemption from both ethical review and informed consent requirements.

Differential lncRNA Identification and Risk Model Construction

Utilizing the ConsensusClusterPlus package, a consistency matrix was constructed through consistency clustering, and the samples were classified based on specific parameters. The clustering algorithm was set to “km” with a distance of “euclidean”. The process was repeated 500 times with an 80% sampling ratio each time to ensure clustering reliability. For ConsensusClusterPlus analysis, the optimal number of clusters (k) was selected based on the cumulative distribution function (CDF) curve and delta area plot. The most stable clustering was observed when k = 2.

Differential lncRNA Identification

Differential analysis between C1 and C2 subtypes was conducted using the limma package with an FDR threshold of 0.05. Subsequently, the survival R package was employed to perform univariate Cox proportional hazard regression on the differential genes, considering a significance level of p < 0.05. For LASSO-Cox regression, the lambda parameter was determined using 10-fold cross-validation to minimize partial likelihood deviance, and the value of lambda.min was selected. Stepwise multivariate regression analysis further reduced the gene set.

Prognostic Model Construction and Validation

LASSO Cox regression analysis was utilized to reduce the number of genes with key genes and correlation coefficients determined through stepwise regression. The risk score for each patient was calculated using a specific formula. The optimal threshold for dividing high and low-risk groups was determined using the survminer package. Kaplan-Meier and ROC analyses were performed to evaluate the prognostic classification of RiskScore using the R software package timeROC.

Immune Infiltration and Therapy Response Prediction

Various packages like GSVA, ESTIMATE, CIBERSORT, TIDE, and pRRophetic were utilized for immune infiltration evaluation and immunotherapy prediction.²² The Immunophenoscore (IPS) was calculated, and the sensitivity of chemotherapy drugs was predicted.

Gene Set Enrichment Analysis (GSEA)

GSEA was employed to analyze different biological processes across molecular subtypes using the HALLMARK pathway gene set downloaded from the molecular feature database (https://www.gseamsigdb.org/gsea/msigdb/).

Cell Culture and Real-Time Fluorescence Quantitative PCR (RT-qPCR)

Human HCC cell line Li-7 (RRID:CVCL_3840) was provided by the stem cell bank of the Chinese Academy of Sciences. Cells were cultured in 1640 (Gibco, USA) medium containing 10% fetal bovine serum at 37 °C and 5% CO₂. The number of 1×10⁶ cells was placed in an ultra-low adsorption 6-well plate and cultured for 24 h under hypoxia conditions containing 1% O₂, 5% CO₂, and 37 °C. All experiments were divided into 3 replicates. Total RNA was extracted using RNeasy Mini Kit (Magen, China) and cDNA was synthesized using PrimeScript TM RT Master Mix (Takara, China). The primers were designed and synthesized by servicebio. RT-qPCR was performed using TB Green^® Premix Ex Taq TM II and LightCycler 480 System. The results show that it is 2^−ΔΔCt. The mRNA expression of 5 lncRNAs was randomly detected, and GAPDH was selected as the internal reference gene. All experiments were divided into 3 replicates. The primer sequence is detailed in Table 1.

Table 1 Primers Used for RT-qPCR

Western Blotting

The cells in the six-well plate were lysed using RIPA lysis buffer and protease inhibitor (PMSF) to extract total protein. The lysed cells were centrifuged at 14,000 rpm for 15 min and the supernatant containing the total protein was quantified by the BCA protein assay kit (Beyotime, Shanghai, China). The 30 µg protein was used for 12% SDS-PAGE electrophoresis to separate the protein, and then the protein was transferred to the PVDF membrane. After blocking with 5% skim milk for 1 h, the membrane was incubated with the primary antibody at 4 °C overnight. After washing with TBST buffer, the second antibody coupled with horseradish peroxidase was added to the membrane and incubated for 40 min. The protein bands were displayed using ECL reagents and analyzed using ImageJ software. The antibodies were as followed: β-actin (Santa Cruz Biotechnology, 1:1000), HIF-1α (Cell Signaling Technology, 1:1000).

Short Interfering RNA (siRNA) Transfection

Li-7 cells were transiently transfected with target-specific siRNA or negative control siRNA (siNC). All individual siRNAs were designed and synthesized by Sangon Biotech. The cells were cultured in six-well plates until they reached 60–70% confluence and then transfected using RNA TransMate reagent (Sangon Biotech). Cells were harvested 48 h post-transfection. The sequence information of siRNA is shown in Table 2.

Table 2 The Sequence Information of siRNA

Flow CytoMetry

Apoptosis was assessed using the Annexin V-APC/DAPI Apoptosis Kit (Elabscience^®, Wuhan, China). The cell quantity and culture conditions are as described in Cell Culture. Harvest the cells and centrifuge at 300 ×g for 5 min. Remove the supernatant, wash the cells once with PBS, and centrifuge again to discard the wash buffer. Resuspend the cells in 100 μL of diluted 1× Annexin V Binding Buffer. Add 2.5 μL of Annexin V-APC Reagent and 2.5 μL of DAPI Reagent (25 μg/mL). Mix thoroughly and incubate at room temperature in the dark for 15 min. Finally, add 400 μL of diluted 1× Annexin V Binding Buffer, mix gently, and analyze the samples using flow cytometry.

Statistical Analysis

All statistical data were analyzed using R language (version 3.6.0). Continuous variables such as gene expression, immune scores, and pathway enrichment levels were compared using the Wilcoxon rank-sum test. Differences in categorical clinical characteristics between groups were evaluated using the chi-square test. Spearman correlation analysis was employed to assess associations between risk scores and immune infiltration or pathway activity. RT-qPCR and flow cytometry data are presented as mean ± standard deviation, and comparisons among multiple groups were conducted using one-way analysis of variance (ANOVA). All tests were two-sided, and a p < 0.05 was considered statistically significant.

Results

Identification of Prognostic Hypoxia- and Anoikis-Related lncRNAs and Classification of HCC Molecular Subtypes

To explore the role of hypoxia- and anoikis-related lncRNAs in HCC, we integrated data from the GSE103581 and GSE188608 datasets, identifying 154 lncRNAs significantly associated with overall survival in the TCGA-LIHC cohort (p < 0.05; Supplementary Table 1). Among them, 61 lncRNAs were differentially expressed between tumor and adjacent normal tissues (Supplementary Figure 1A), and 49 were further validated to be prognostically relevant (lncRNA_cox.csv). The intersection of these datasets yielded 25 lncRNAs (Supplementary Figure 1B), among which LINC01018 and LINC01554 were more highly expressed in normal tissues, while the remaining lncRNAs were upregulated in tumor samples (Supplementary Figure 1C).

Based on the expression profiles of these 25 lncRNAs, consensus clustering analysis was performed using the TCGA-LIHC dataset. Two robust molecular subtypes, C1 and C2, were identified (Figure 2A and B). Survival analysis revealed a significantly worse prognosis in the C1 subtype compared to C2 (Figure 2C). Further characterization using the six established immune subtypes (C1–C6) demonstrated that the majority of HCC samples in both molecular subtypes corresponded to immune subtypes C3 (inflammatory) and C4 (lymphocyte-depleted), with minimal overlap with C5 (immunologically quiet) and C6 (TGF-β dominant) (Figure 2D). Notably, C2 contained a higher proportion of patients with aggressive immune subtypes (C1 and C2), consistent with its poorer immune-associated survival outcomes (Figure 2E). These findings highlight the potential of hypoxia- and anoikis-related lncRNAs not only as prognostic markers but also as classifiers of HCC molecular subtypes with distinct immune landscapes and clinical outcomes.

Figure 2 Molecular subtype construction and prognosis analysis. (A) TCGA-LIHC sample clustering heat map. (B) PCA of TCGA molecular subtypes. (C) Survival analysis between TCGA subtypes. (D) Comparison of the distribution of immune subtypes between different molecular subtypes. (E) Survival curve of immune subtypes.

Integrated Genomic and Immunological Characterization of C1 and C2 Subtypes Reveals Distinct Tumor Biology and Immunotherapy Responses

To further elucidate the biological differences between the C1 and C2 subtypes, we investigated their genomic alterations, somatic mutations, immune microenvironment profiles, and pathway enrichment.²³ Genomic instability was more pronounced in the C1 subtype, which exhibited significantly higher levels of fraction genome altered, number of segments, and homologous recombination deficiency scores (Figure 3A). Fisher’s exact test identified subtype-specific somatic mutations (p < 0.01), revealing that C1 had higher mutation frequencies in TP53, DMD, TG, and GREB1, whereas C2 was enriched for mutations in BIRC6, DOCK8, HERC1, IL6ST, CREBBP, and OR2J3 (Figure 3B and C).

Figure 3 Genomic characteristics and somatic mutations among different subtypes. (A) Genome feature score between C1 and C2 subtypes. (B and C) Forest map and waterfall map of differential mutation genes between C1 and C2 subtypes. Notes: (C) X-axis shows gene names. Each cell shows mutation frequency (%), with colors or symbols representing mutation types.

We next examined the immune landscape of these subtypes. ESTIMATE analysis showed that the C1 subtype had elevated stromal and immune scores, indicating increased immune and matrix component infiltration (Figure 4A). CIBERSORT analysis revealed that C1 harbored higher proportions of immunosuppressive cells, including regulatory T cells (Tregs), M0 macrophages, and memory B cells (Figure 4B). In contrast, single-sample GSEA (ssGSEA) demonstrated overall enhanced immune activation in C1, including increased infiltration of CD4+ and CD8+ T cells, B cells, NK cells, dendritic cells, and macrophages. Despite this heightened immune cell presence, the elevated myeloid-derived suppressor cell (MDSC) score in C1 suggests a coexisting immunosuppressive milieu, potentially contributing to immune evasion. Meanwhile, C2 showed modest immune activity with relatively higher scores for activated dendritic cells (Figure 4C). Integrative comparison with immune-related genomic signatures reported in HCC literature indicated that the C1 subtype scored significantly higher in proliferation, TGF-β response, and aneuploidy, supporting a more aggressive and genomically unstable phenotype (Figure 4D). Immunotherapy sensitivity prediction revealed higher Immunophenoscore (IPS) and lower TIDE scores in the C2 subtype, suggesting greater potential responsiveness to immune checkpoint blockade in these patients (Figure 4D). Finally, we compared the differences in activation pathways between C1 and C2 subtypes. GSEA pathway analysis showed that C1 was enriched in oncogenic and EMT-related pathways, including E2F targets, G2M checkpoint, and epithelial-mesenchymal transition, while C2 was associated with metabolic pathways, particularly bile acid metabolism (Figure 4E).

Figure 4 Analysis of immune microenvironment of two subtypes. (A) ESTIMATE immune score difference between subtypes. (B) CIBERSORT immune infiltration difference between subtypes. (C) 28 immune score differences between subtypes. (D) Proliferation, TGF-beta Response, Aneuploidy score, and immunotherapy sensitivity comparison between subtypes. (E) Differences in pathway activity between subtypes. Notes: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Construction and Validation of a Prognostic 9-lncRNA Risk Model

A total of 61 differentially expressed lncRNAs were identified between the C1 and C2 subtypes (Figure 5A). These candidates were first subjected to univariate Cox regression to screen for prognosis-related genes, followed by LASSO Cox regression to reduce overfitting and refine the model (Figure 5B and C). Subsequently, stepwise multivariate Cox regression further narrowed the list, and nine lncRNAs were ultimately identified as independent prognostic factors: LINC01554, LINC01134, LINC00661, LINC01096, MIAT, NBAT1, PICSAR, FIRRE, and LINC01139 (Figure 5D). The final risk model was constructed based on their expression and corresponding coefficients as follows: Risk score = (−0.044 * LINC01554) + (0.168 * LINC01134) + (0.053 * LINC00661) + (0.056 * LINC01096) + (−0.258 * MIAT) + (0.067 * NBAT1) + (0.074 * PICSAR) + (0.093 * FIRRE) + (0.028 * LINC01139).

Figure 5 Nine lncRNAs were identified as key genes affecting prognosis. (A) Differential lncRNA identification between C1 and C2 subtypes in TCGA. (B) Differential lncRNA prognostic forest map. (C) LASSO narrows the gene range. (D) Multi-factor forest map of characteristic genes. Note: The volcano map threshold is adj.p <0.05 and |log2FC|> 1.

Based on this formula, risk scores were calculated for each patient, and individuals were stratified into high- and low-risk groups. Kaplan-Meier survival analysis revealed that patients in the high-risk group had significantly worse overall survival than those in the low-risk group (Figure 6A). The model also showed strong predictive accuracy, with a high area under the ROC curve (AUC) (Figure 6B). Expression profiling demonstrated that LINC01554 and MIAT were predominantly expressed in the low-risk group, whereas the other seven lncRNAs were significantly upregulated in the high-risk group (Figure 6C). The prognostic value of this model was further validated in the independent GSE43619 cohort, with similar survival stratification and predictive performance (Figure 6D–F).

Figure 6 A prognostic risk model was constructed based on 9-lncRNA. (A–C) ROC curve of risk model, KM survival curve and heat map of 9-lncRNA expression between risk groups in TCGA cohort. (D–F) ROC curve of risk model, KM survival curve and 9-lncRNA expression heat map between risk groups in GSE43619 cohort. Notes: (A, D) The X-axis represents the follow-up time (years), and the Y-axis indicates overall survival probability.

Multi-Dimensional Evaluation of the 9-lncRNA Risk Model: Prognostic Value, Immune Infiltration, and Drug Response

To evaluate the clinical relevance of the 9-lncRNA-based risk score, we compared the clinicopathological features between high- and low-risk groups in the TCGA cohort. As tumor grade and AJCC stage increased, the corresponding risk score also significantly increased, indicating a strong association between molecular risk and disease severity (Figure 7A). Univariate and multivariate Cox regression analyses identified both RiskScore and AJCC stage as independent prognostic factors for overall survival (Figure 7B and C). A combined nomogram integrating RiskScore and AJCC stage demonstrated that RiskScore had the greatest impact on survival prediction (Figure 7D). The calibration curve showed strong agreement between predicted and observed survival at 1, 3, and 5 years (Figure 7E), and decision curve analysis (DCA) confirmed that the nomogram and RiskScore provided greater net clinical benefit than traditional clinical indicators (Figure 7F).

Figure 7 RiskScore combined with clinicopathological features to improve prognostic model and survival prediction. (A) Riskscore difference between clinical features in TCGA. (B and C) Riskscore and clinical characteristics of single factor and multivariate results. (D) Riskscore combined with AJCC Stage to establish a nomogram. (E and F) Calibration and Decision Curves for Nomogram.

To explore immune landscape differences between risk groups, we used ESTIMATE and found that the low-risk group had significantly higher StromalScore, ImmuneScore, and ESTIMATEScore, along with lower tumor purity, suggesting a more active immune microenvironment (Figure 8A and B). Further correlation analysis using CIBERSORT indicated that RiskScore was negatively associated with CD8⁺ T cells and M1 macrophages, but positively correlated with immunosuppressive cells such as Tregs and M0 macrophages (Figure 8C). Moreover, the low-risk group exhibited higher Immunophenoscore (IPS) and lower Tumor Immune Dysfunction and Exclusion (TIDE) scores, suggesting a greater potential for response to immunotherapy (Figure 8D and E). RiskScore also showed a significant correlation with key immune-related signatures, including IFN-γ response, TGF-β response, and aneuploidy score, underscoring its impact on the tumor immune microenvironment (Figure 8F).

Figure 8 Difference analysis of tumor microenvironment in TCGA risk group. (A) ESTIMATE score between TCGA risk groups. (B) Tumor purity difference between TCGA risk groups. (C) Correlation between Riskscore and CIBERSOER immune score. (D and E) Comparison of immunotherapy sensitivity between TCGA risk groups. (F) Correlation analysis between Riskscore and IFN-gamma Response, TGF-beta Response, Aneuploidy score. Notes: *p < 0.05, **p < 0.01, ***p < 0.001.

To assess potential chemotherapeutic responsiveness, we predicted drug sensitivity across risk groups using pRRophetic. Notably, BI-2536, GNF-2, WH-4-023, Vinorelbine, and A-443654 were predicted to be more effective in the high-risk group, while Roscovitine, HG-6-64-1, KIN001-135, Phenformin, and DMOG were more suitable for the low-risk group (Figure 9A). Finally, pathway analysis using ssGSEA revealed that RiskScore positively correlated with proliferation-related pathways (eg, E2F targets, G2M checkpoint), and negatively correlated with metabolism-related pathways (eg, bile acid metabolism, fatty acid metabolism) (Figure 9B). These findings suggest that the 9-lncRNA RiskScore not only reflects clinical aggressiveness and immune evasion, but also informs therapeutic stratification and targeted treatment decisions.

Figure 9 Drug sensitivity and functional enrichment analysis in the prognostic model. (A) Drug sensitivity difference between risk groups. (B) Riskscore and differential pathway correlation.

The Expression of Five lncRNAs Was Verified by RT-qPCR and Western Blotting

In order to verify the expression of five lncRNAs in the model, the liver cancer cell line Li-7 was inoculated into an ultra-low adsorption culture plate and cultured under hypoxia conditions for 24 hours to establish a hypoxia-anoikis model (Figure 10A). The expression of LINC01554, FIRRE, LINC01139, LINC01134 and NBAT1 was detected by RT-qPCR. The results showed that the expression of LINC01554 decreased, while the expression of FIRRE, LINC01139, LINC01134 and NBAT1 was lower (Figure 10B). Subsequently, siRNA was employed to knock down the expression of LINC01554 and LINC01139 in Li-7 cells, with apoptosis evaluated under a hypoxia-anoikis model using flow cytometry. Compared to the control group, the expression levels of LINC01554 and LINC01139 were significantly reduced (Figure 10C). Notably, the proportion of apoptotic cells was decreased in the si-LINC01554 group and increased in the si-LINC01139 group relative to the si-NC group (Figure 10D).

Figure 10 Western blotting and RT-qPCR were used to verify the LncRNA in the cell hypoxia and anoikis model. (A) Hypoxia of liver cancer cell line Li-7 was verified by Western blotting. (B) The relative expression of LINC01554, FIRRE, LINC01139, LINC01134 and NBAT1 mRNA. (C)The LINC01554 and LINC01139 interference efficiency in Li-7 cells was determined by RT-qPCR. (D) The percentage of apoptotic cells in si-LINC01554 and si-LINC01139 was determined by flow cytometry. The experimental data were expressed as the mean ± SD of the three independent experiments, and the asterisks indicated p values (** p < 0.01,*** p < 0.001,**** p < 0.0001).

Discussion

HCC is one of the leading causes of cancer-related deaths globally, with metastasis being a major contributor to patient mortality.²⁴ Due to the high rates of recurrence and metastasis, the prognosis for HCC patients after chemotherapy or drug treatment remains poor.²⁵ While some biomarkers assist in decision-making and guiding HCC treatment, they are still limited.²⁶ Alpha-fetoprotein (AFP) is an important diagnostic biomarker for HCC. However, over 30% of HCC patients exhibit AFP negativity, highlighting the critical need for new biomarkers.²⁷ LncRNAs, due to their tissue specificity, stability, and significant roles in gene regulatory networks, offer advantages as therapeutic and predictive biomarkers.²⁸ The lncRNA MIR210HG can promote HCC tumorigenesis and angiogenesis by upregulating the expression of mRNA PFKFB4 and SPAG4, effectively predicting the prognosis of HCC patients. It can provide important clinical references for evaluating patient recurrence and metastasis risks.²⁹ The discovery and application of more lncRNA biomarkers will significantly enhance the prediction and diagnostic capabilities of HCC metastasis, providing new avenues for developing personalized treatment strategies.

Hypoxia and anoikis are common stress factors in the tumor microenvironment that impact tumor progression and metastasis by regulating gene expression and cell signaling pathways. The hypoxia microenvironment induces significant changes in the expression of numerous lncRNAs, impacting the behavior of HCC cells. HABON is transcriptionally activated by HIF-1α under hypoxia conditions to facilitate the transcriptional activation of BNIP3, leading to elevated BNIP3 expression levels and promoting the growth, proliferation, and clone formation of HCC cells under hypoxia conditions.³⁰ Cancer recurrence and metastasis represent a multifaceted process involving various steps and factors, wherein evading anoikis serves as a pivotal stage.³¹ HCC cells thwart anoikis and bolster metastasis through diverse molecular mechanisms, including integrin signaling, oxidative stress, and Epithelial-Mesenchymal Transition (EMT).^32–34 Notably, the collaboration between integrin β4 and the epidermal growth factor receptor (EGFR) enhances HCC resistance to anoikis by activating the FAK-AKT signaling pathway.³⁵ LncRNA plays a crucial role in the regulation of anoikis. For example, studies have shown that the LncRNA FOXD2-AS1/miR7/TERT pathway can enhance the survival rate and anchorage-independent growth of thyroid cancer cells.³⁶ LncRNA HOTAIR plays a crucial role in EMT by regulating the expression and activation of c-Met and its membrane co-localization partner Caveolin-1, as well as membrane organization, thereby helping HCC cells produce anoikis resistance and evade tumor immunity.³⁷ Our data indicate that certain lncRNAs undergo changes under hypoxia and anchorage-independent conditions, potentially serving as prognostic biomarkers for HCC.

In this study, through a comprehensive analysis of hypoxia- and anoikis-related lncRNAs, HCC patients were categorized into two molecular subtypes using cluster analysis. The proportion of subtype C1, characterized by a proliferative phenotype, and C2 immune subtypes exceeded that of subtype C2. HCC was classified into proliferative and non-proliferative subtypes, with the former displaying high proliferation rates, chromosomal instability, and activation of the Akt/mTOR signaling pathway.³⁸ The proliferative subtype correlated with immune subtypes C1 and C3, while C2 was linked to the non-proliferative subtype. Subtype C1 was distinguished by poor differentiation, elevated tumor grade, presence of macrovascular invasion, increased proliferation markers (PLK1, MKI67), and overexpression of stem cell genes (EPCAM and AFP).³⁹ This study revealed that tumor cells of C1 subtype tended to accumulate mutations, showcasing heightened heterogeneity and malignancy, resulting in a poorer patient prognosis. These findings suggest that the C1 subtype exhibits a stronger immune evasive capability and lower sensitivity to immunotherapy, whereas patients with the C2 subtype demonstrate a relatively improved prognosis.

Concerning immunotherapy, tumors are categorized into three types: immune-desert, immune-excluded, and immune-inflamed, determined by the presence and activity of immune cells within the tumor microenvironment.⁴⁰ Immune rejection primarily manifests as an immunosuppressive state, featuring ineffective immune cell infiltration that hinders T cells from reaching the core of the tumor due to various immunosuppressive factors.⁴¹ The immune-inflamed type denotes an active immune response characterized by substantial infiltration of T cells and other immune cells in the tumor, alongside high expression levels of inflammation-related cytokines.⁴² Our risk scoring model, based on nine lncRNAs, further confirmed the prognostic value of these lncRNAs in HCC. We noted that the high-risk group, particularly the C1 subtype, exhibited more immunosuppressive elements in the tumor microenvironment, including increased expression of regulatory T cells (Tregs), inactivated M0 macrophages, and MDSC. This aligns with their lower responsiveness to immunotherapy, suggesting a potentially limited reaction to current immune checkpoint inhibitors (ICI). This corresponds to the characteristics of the “immune-excluded” subtype in HCC immunotyping, where tumors typically exhibit a highly immunosuppressive microenvironment that hinders immune cell infiltration and cytotoxic activity, and express high levels of immunosuppressive molecules such as PD-L1 and CTLA-4.⁴³ Notably, activated CD8+ T and NK cells coexist with immunosuppressive cells, suggesting a possible “immune-exhausted” state that may limit effector function.⁴⁴ This implies that immunosuppression could be a barrier to immunotherapy, highlighting the potential need for combined targeting strategies. Conversely, the low-risk group, primarily the C2 subtype, displayed reduced immunosuppressive factors and heightened immune cell activity, such as activated CD8+ T cells and memory CD4+ T cells, in line with the profiles of “immunoinflammatory” HCC. Immunoinflammatory HCC typically exhibits increased immune activity and enhanced sensitivity to ICI treatment. Consequently, patients with the C2 subtype show a more favorable prognosis and a more positive response to immunotherapy. Conversely, the high-risk group may demonstrate a diminished response to existing immune checkpoint inhibitors due to its specific traits. Potential therapeutic targets include PD-1/PD-L1, CTLA-4, TGF-β, and VEGF pathways, which, when targeted, can alleviate T cell suppression, improve the tumor microenvironment, and boost the anti-tumor immune response.⁴⁵ Different immunophenotypes of HCC display diverse responses to immune checkpoint inhibitors like PD-1/PD-L1 inhibitors, highlighting the need to explore combined treatment strategies to enhance outcomes.⁴⁶ For instance, the combination therapy of atezolizumab (anti-PD-L1) and bevacizumab (anti-VEGF) has received approval as a novel first-line treatment, significantly enhancing survival rates.⁴⁷ LncRNA holds substantial promise in immune combination therapy by regulating immune checkpoints, acting as a predictive biomarker, offering insights into therapeutic efficacy, and serving as a target or tool in combined therapy.^48–50 For example, lncRNA UCA1 augments the anti-tumor effect of PD-1 inhibitors by suppressing miR-204-5p and boosting PD-L1 expression.⁵¹ LncRNA can play a crucial role as a target or tool in collaboration with other treatments. As an illustration, si-PROX1-AS1 interacts with miR-520d to modulate PD-L1, promoting colorectal cancer (CRC) cell growth, spread, and evasion of the immune response.⁵² These lncRNAs contribute to enhancing the effectiveness of immunotherapy and possess significant potential in combined immunotherapy.

In this study, a prognostic model was constructed based on nine hypoxia- and anoikis-related lncRNA (LINC01554, MIAT, FIRRE, LINC01139, LINC01096, PICSAR, LINC01134, NBAT1, LINC00661) genes, which revealed the important role of HCC in prognosis and tumor immunophenotype, as well as the metastasis and progression of HCC, and could be used as a reliable biomarker for predicting the prognosis and immunotherapy response of HCC. This study identifies hypoxia- and anoikis-related lncRNA subtypes and constructs a prognostic model, providing new insight into the molecular classification and treatment stratification of HCC.

Among the nine hypoxia- and anoikis-related lncRNAs, LINC01554 and LINC01139 were selected for focused experimental validation. Our results showed that under hypoxia and anoikis conditions, knockdown of LINC01554 inhibited apoptosis, suggesting its role as a tumor suppressor. In contrast, knockdown of LINC01139 significantly increased apoptosis, indicating that it promotes tumor cell survival. Previous studies have reported that LINC01554 suppresses HCC progression by regulating the miR-148b-3p/EIF4E3 axis, while LINC01139 facilitates tumor development through the miR-30/MYBL2 axis.^53,54 Moreover, LINC01139 is involved in modulating glucose metabolism disturbances, remodeling the tumor microenvironment, and enhancing immunotherapy efficacy.⁵⁵ These lncRNAs may be regulated by the hypoxia-anoikis tumor microenvironment; however, their precise functions in HCC remain to be fully elucidated. The exact upstream regulatory mechanisms and downstream signaling pathways of these lncRNAs require further in-depth functional studies, representing important directions for future research.

Nonetheless, this study has several limitations. Although the prognostic value of our model was validated in both TCGA and GSE43619 cohorts, it has yet to be confirmed in larger, prospective, and multi-center clinical datasets to establish its true clinical utility. Moreover, while RT-qPCR was used to verify lncRNA expression, the precise mechanisms by which these lncRNAs regulate gene expression and cellular behavior remain unclear and require further functional investigation. Although preliminary apoptosis assays under hypoxia–anoikis conditions support their functional relevance, more comprehensive validations—including proliferation, migration, invasion assays, rescue experiments, and exploration of downstream pathways such as PI3K/AKT and EMT—are necessary. Future studies should also integrate advanced techniques like single-cell sequencing and incorporate clinical samples to fully elucidate the roles of these lncRNAs in HCC progression and their potential clinical applications.

Ethical Statement

In accordance with Article 32 of the Ethical Review Measures for Life Sciences and Medical Research Involving Humans (China, 2023), secondary research using fully anonymized data in non-interventional settings is exempt from both ethical review and informed consent requirements. This study did not involve any interaction with human participants, collection of biological samples, or implementation of invasive procedures. Therefore, it meets the current regulatory criteria for exemption from institutional ethical approval and informed consent.

Acknowledgments

Special thanks to Guangxi Key Laboratory of Traditional Chinese Medicine and Preventive Medicine for supporting this study.

Disclosure

There is no conflict of interest in all authors.

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