With the introduction of all-oral direct-actingantiviral therapy, a substantial breakthrough has been made in chronic HCV infection treatment during the past decades (1). However, due to HCV's significant heterogeneity and high variability, virus reinfection following successful treatment remains an important public health problem (2). The HCV pathogenesis and progression are complex and interact with its biological characteristics, environmental behavior factors, host immunity, and genetic background.

The activation of NF-κB and NF-κB-dependent inflammatory pathways are important to chronic HCV infection and its related cirrhosis and HCC. The NF-κB ligand (RANKL) receptor activator, a 316-amino acid transmembrane protein, is highly expressed in different immune cells including T or dendritic cells. RANKL can be induced by inflammatory factors such as interleukin 1, tumor necrosis factor α, and transforming growth factor β (3). Besides, RANK and osteoprotegerin (OPG) are RANKL receptors, and the RANKL/RANK/OPG pathway is important for cellular immune responses such as cell death and proliferation, inflammation, and immunity (4). The communication pathways mediated by TNFSF/TNFRSF are essential for numerous developmental, homeostatic, and stimulus-responsive processes. Both innate and adaptive immune cells are controlled by TNFSF/TNFRSF members in a manner that is crucial for the coordination of various mechanisms driving either co-stimulation or co-inhibition of the immune response (5). Different cellular immune responses can be triggered by genetic differences between different individuals. During the past decades, many studies have identified that immune cytokines SNPs were significantly associated with HCV spontaneous clearance and virological response, except for viral and environmental factors (6–9). Studies have shown that multiple TNFSF and TNFRSF gene SNPs are related to autoimmune diseases, suggesting that these SNPs play an important role in immunity. rs8086340 and rs2073618 are closely related to the occurrence of rheumatoid arthritis (10), and rs9525641 may affect the susceptibility and severity of AS disease (11). However, no research has addressed TNFSF and TNFRSF genetic polymorphisms' impacts on HCV-related chronic liver diseases. Considering that China has the largest number of HCV infections (about 10 million patients), we examined the relationships between OPG-RANKL-RANL pathway genes SNPs rs9525641, rs2073618, and rs8686340, and HCV infection outcomes in a high-risk Chinese population.

The RANKL message is detected in the peripheral lymph nodes and bone marrow, thymus, spleen, Peyer's patches, brain, heart, skin, skeletal muscle, kidney, liver, lung, and mammary tissues (12). The RANKL/RANK system has been shown to play a critical role in the immune system, including lymph-node development, lymphocyte differentiation, dendritic cell survival, and T-cell activation and tolerance induction (13). RANKL can regulate lymph-node organogenesis, T- and B-lymphocyte development, and osteoclast differentiation. Some studies also indicated that RANKL regulates the thymus microenvironment by autoimmune regulators expression activation (14). Additionally, at the molecular level, RANK interacts with RANKL to activate the transcription factor NF-κB along with TNF receptor-related factor family signaling molecules (15). Considering that the NF-κB function is related to RANKL/RANK, and NF-κB has been linked to chronic hepatitis C (16–18), we hyphothesized that the RANKL/RANK pathway polymorphisms would affect the HCV infection outcome.

RANKL, RANK, and OPG are encoded by TNFSF11 (gene map locus 13q14), TNFRSF11A (gene map locus 18q22.1), and TNFRSF11B (gene map locus 8q24), respectively (19). The TNFSF11 gene structure is highly conserved among mammals, consisting of five exons that span 33.9 kb in humans (10). SNPs located near TNFSF11, TNFRSF11A, and TNFRSF11B have been reported to be closely associated with Paget's disease (20), osteoporotic fractures (21), cardiovascular diseases (22), ankylosing spondylitis (11), and breast (23), and esophageal cancers (24). In this study, we showed that subjects who carried the rs9525641-C allele were more likely to develop HCV chronicity than those with the rs9525641-T allele. Furthermore, in the stratification analyses based on age, gender, and high-risk population, we found that the rs9525641-C allele was associated with HCV chronicity among elders, females, and persons with ALT and AST <40 U/L. Interestingly, RANKL can cause various degenerative bone diseases, such as rheumatoid arthritis and osteoporosis. These diseases are mostly female. Our research shows that RANKL is related to the chronicity of HCV in women. This suggests to some extent the combined effect of gender factors and the RANKL system in disease progression, which is worthy of further discussion. However, the heterogeneity test showed no significant heterogeneity in any pair-wise comparison (p > 0.05), indicating that these variables did not materially affect the results.

Although rs9525641 is located in the TNFSF11 intronic region, this variant might play an important role in gene transcription regulation or might be in linkage disequilibrium with other functional SNPs, such as rs17458177, rs1325799, rs17536328, rs7984870, rs9533155, and rs3742257. We also calculated the degree of linkage between the candidate SNPs, r² < 0.1 indicates that there is no linkage disequilibrium among these three SNPs. The bioinformatics analysis indicated that these variants could regulate gene transcription, mRNA export, and protein translational efficiency. In the present study, we found two possible TNFSF11 biological processes using the STRING database website prediction (https://string-db.org/cgi/network.pl?taskId=kGEafB5GcPa7:) extracellular signal-regulated kinase 1 (ERK1) and ERK2 cascade positive regulation via TNFSF11-mediated signaling; and the tumor necrosis factor-mediated signaling pathway. It has been reported that the HCV non-enveloped particles' intriguing cellular internalization properties can activate the ERK1/2 pathway that could be important in the HCV life cycle and infection pathogenesis (25). Additionally, Fletcher et al. reported that several TNF superfamily members – including TNF-α, TNF-β, TWEAK, and LIGHT – can promote HCV entry via NF-κB-mediated activation of myosin light chain kinase and tight junctions disruption (26). Therefore, TNFSF11 may be involved in these pathways and contribute to the hepatitis C chronic process. However, these hypotheses are based on computer simulations and functional evaluations using biological assays. Thus, it should be warranted in future studies.

RANK is an intrinsic hematopoietic cell surface receptor that stimulates NF-κB receptor activation, plays a central role in T and dendritic cells, and promotes lymph node development (27)^. Previously, several studies have found that the TNFRSF11 rs8680340 was closely related to the anti-citrullinated peptide antibody and age at natural menopause (28). Additionally, the TNFRSF11B rs2073618 was significantly associated with Type 2 Diabetes (29), rheumatoid arthritis (30), and volumetric bone mineral density (31). However, in this study, no significant association was observed between TNFRSF11A rs8086340, TNFRSF11B rs2073618, and HCV infection outcome.

Our study also has some limitations. First, this study was performed with the Chinese Han population, requiring further reproduction in different ethnic populations. Second, we did not collect immune markers data in the RANKL/RANK pathway and could not check the association between immune markers data and the target SNPs. Third, we did not collect complete virus subtypes and viral load data and could not check the association between virus data and the target SNPs. Hence, we should improve the collection of this part of the data in the later stagethe and possible immune mechanism requires to be further studied and verified. Furthermore, we did not explore how the functional mechanisms of these SNPs affect HCV chronicity using molecular biology approaches. Then, functional studies are required in the future to explore how these polymorphisms impact chronic HCV infection.

Overall, our findings suggested that the rs9525641 TNFSF11 polymorphism might affect HCV chronicity in a high-risk Chinese population. Larger well-designed epidemiological studies with ethnically diverse populations and functional evaluations are warranted to confirm these findings before the effect of these variants can be fully and accurately evaluated.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

The studies involving human participants were reviewed and approved by the Ethics Committee of Nanjing Medical University. The patients/participants provided their written informed consent to participate in this study.

SY and R-BY: conceptualization. Y-QH: formal analysis, investigation, and writing–original draft. PH: funding acquisition and methodology. Y-DW and J-JW: resources. J-JW and FZ: software. SY: supervision. Y-QH and X-YY: writing–review and editing. All authors contributed to the article and approved the submitted version.

This study was supported in part by National Natural Science Foundation of China (no. 81703273), Natural Science Foundation of Jiangsu Province (no. BK20171054), Natural Science Foundation of Yunnan Province (no. 2019FA005), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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