Associations Of Single Nucleotide Polymorphisms Of The Immunoregulatory And Complement System Related Genes With Overall Survival Of HBV-associated Hepatocellular Carcinoma After Hepatectomy

Primary liver cancer is one of the most common malignant tumors around the world,and an estimated 830,000 people died from liver cancer worldwide in2020,with accounting for 47.1%occurred in China.Hepatocellular carcinoma（HCC）accounts for 85%-90%of all liver cancer cases,and nearly 95%of HCC cases have a history of HBV infection in China.Although the clinical treatment effect of HCC has made great progress in recent years,the overall survival of HCC patients is still not ideal.Clinically,for HCC patients with the same clinical characteristics,even if they receive the same treatment plan,the prognosis of each is very different,suggesting that genetic factors may play an important role.Therefore,exploring the influence and its mechanism of genetic factors on the progression of HCC will help us better assess and predict the prognosis of HCC patients.The immune system plays an important role in the occurrence and development of HCC,and more attention focus on the immune regulation and its mechanism now.A numbers of studies have found that the abnormal expression of immune-related genes was associated with the prognosis of tumor patients,which may be related to its involvement in regulating the level of immune cells infiltration level in the tumor microenvironment.The imbalance of the immune microenvironment can promote immune tolerance and immune escape through a variety of ways,causing imbalance of immune regulation of the liver,thus promoting the occurrence and development of HCC.The complement system is a group of proteins that have enzymatic activity in human blood and tissue fluid after activation.In tumor immunotherapy,the complement system can mediate antibody-based immunotherapy to lyse tumor cells.Studies have found that abnormal activation of the complement system in the tumor immune microenvironment can cause tumor immune infiltrating cells to release inflammatory factors,inhibiting the activation of T cells,and then promoting tumor progression.As one of the most common type of human heritable variation,SNP is an important factor in determining individual genetic susceptibility,sensitivity to drugs,the prognosis of individuals with diseases.In recent years,genome-wide association studies（GWAS）have discovered a large number of SNPs associated with the genetic susceptibility and prognosis of tumors.However,most SNPs are located in the non-coding region or gene desert region,and their biological functions are not clear.In the post-GWAS research era,research strategy based on biological pathways will explore the GWAS data more comprehensively and deeply.By comprehensively analyzing the cumulative effect between a set of SNPs or genes in the signal pathway and tumors,it can effectively make up for the shortcomings that only the strength of association can be found in the analysis of a single site.This strategy can provide new ideas for studying tumor pathogenesis and prognostic judgment.In the present study,we adopted a cohort study method and toke HBV-associated HCC patients after hepatectomy as subjects to establish the HBV-associated HCC GWAS database in Guangxi,China.Then we systematically explored the associations of SNPs of the immunoregulatory and complement system related genes with overall survival of HBV-associated HCC after hepatectomy.Through using the bioinformatics tools and public databases for function prediction of SNPs and carrying out the molecular biology experiments,we explore preliminary the biological mechanism of SNPs and corresponding genes in the occurrence and development of HCC.The results of this study will provide new ideas for the prognostic judgment of HCC patients and the development of therapeutic targets,as well as an important theoretical basis for the precise prevention and precise treatment of HCC in the future.Objective:To explore the associations between genetic variants of immunoregulatory and complement system related genes and overall survival（OS）in patients with HBV-associated HCC after hepatectomy,and carry out the biological functions of significant SNPs and genes.Methods:1.In this study,a cohort study was used to study HBV-associated HCC patients after hepatectomy.Genotyping was performed on DNA samples of all subjects using genome-wide gene microarray to establish 866HBV-associated HCC GWAS database.2.Multivariate Cox regression model was used to analyze the associations between the postoperative OS of HBV-associated HCC patients with 6593 SNPs of 124 immunoregulatory genes and 7790 SNPs of 103 complement system related genes in two datasets.SNPs that significantly associated with postoperative OS in HBV-associated HCC patients were screened out,and BFDP with a threshold of 0.80 was used for multiple test correction to reduce the incidence of false positives.3.Based on the genotyping data of 103 Chinese Han people in 1000 Genomes Project,Haploview software was used to analyze the linkage disequilibrium of the screened SNPs.Step multivariate regression analysis was used to select SNPs that independently associated with postoperative OS in HBV-associated HCC patients.Bioinformatics tools such as Haploreg V4.1,Regulomed B,SNPinfo,mi RNASNP-V3 and mi RSNP were adopted to annotate the biological functions of the obtained SNPs.4.Multivariate Cox regression model and Kaplan Meier method were used to analyze the influence of selected SNPs on postoperative OS in HBV-associated HCC patients under different genetic models（additive model,dominant model and recessive model）.The Bootstrap method was used to conduct 1000re-sampling to calculate the HR for each time,and Shapiro-Wilk test was used to calculate the degree of fitting and 95%CI of the normal distribution of HR values.5.To evaluate the cumulative effect of SNPs on postoperative OS in HBV-associated HCC patients,SNP-SNP interaction analysis was performed.Risk genotypes of SNPs and clinical factors（age,sex,smoking status,alcohol consumption status,AFP,cirrhosis,tumor thrombus,BCLC stage）were combined to build a prediction model.ROC curve and AUC were calculated to evaluate the predictive ability of the prediction model for postoperative survival of HBV-associated HCC patients.6.e QTL analysis was performed using GTEx database to evaluate the relationship between different genotypes of SNP and m RNA expression levels of corresponding genes.Dual luciferase reporter gene assay was carried out to verify the effect of SNP on gene m RNA expression.7.The m RNA expression profile data of HCC cancer tissue and paracancerous tissue samples from TCGA database and Kaplan-Meier Plotter database were used to detect m RNA expression levels to analyse the difference and their relationship with the prognosis of patients,as well as 78 pairs of HBV-associated HCC patients by real-time quantitative PCR technology.Using data from Cbioportal database and tumor immunity database Timer2.0,we analyzed somatic mutations of related genes in HCC cancer tissues and their relationship with the level of tumor microenvironment immune cell infiltration.Results:I associations between SNPs of the immunoregulatory related genes and overall survival of HBV-associated HCC after hepatectomy1.Through the multivariate Cox regression analysis of the discovery set and the validation set of 6593 SNPs on 124 immune regulation-related genes,we found that 3 potentially functional SNPs（CXADR rs2824339 C>T,COLEC12rs8085897 C>G and SIGLEC6 rs10221508 C>T）were significantly associated with the postoperative OS of HBV associated HCC patients.Among them,CXADR rs2824339 T allele and COLEC12 rs8085897 G allele could reduce the postoperative mortality risk of HBV-associated HCC patients（rs2824339:CT+TT vs.CC:HR=0.73,95%CI=0.60-0.89,P=0.002;rs8085897:CG+GG vs.CC:HR=0.66,95%CI=0.53-0.82,P=0.002）,while the SIGLEC6 rs10221508T allele increases the risk of postoperative death（CT+TT vs.CC:HR=2.73,95%CI=1.34-5.58,P=0.006）.2.In this study,after combining the genotypes of 3 significant SNPs（CXADR rs2824339 CC,COLEC12 rs8085897 CC and SIGLEC6 rs10221508 CT or TT）,we found that the more risk genotypes HBV associated HCC patients carry,the worse the prognosis of patients had,it showed a dose-response effect(P_trend<0.001).Compared with HCC patients who carrying 0 or 1 risk genotype,patients with 2 or 3 risk genotypes have a 1.48 fold increase in postoperative mortality risk（HR=1.48,95%CI=1.22-1.80,P<0.001）.3.The results of ROC curve analysis show that compared with the prediction model constructed using clinical factors alone（including age,gender,smoking status,drinking status,AFP,liver cirrhosis,cancer thrombus,Barcelona staging,etc.）,the predictive model constructed based on the combined risk genotypes and various clinical factors can improve the ability to predict the 3 year OS of patients with HBV associated HCC（3-year survival:72.72%vs.74.09%,P=0.043）.4.In this study,the GTEx database was used to perform the e QTL analysis of significant SNPs and we found that the rs2824339 T allele was associated with increased CXADR m RNA expression levels in 208 liver tissues（P=0.034）.Using bioinformatics tools,such as Regulome DB,Haplo Reg,and SNPinfo,we predict the regulatory mechanism of rs2824339 and found no potential functions.Then,we found that rs422668（T>C）located at CXADR 3’UTR was highly linked to rs2824339（r²>0.8）,and the rs422668 C allele was associated with increased CXADR m RNA expression in 208 liver tissues（P=0.028）.Using bioinformatics tools to predict,such as SNPinfo,mi RNASNP and mir SNP,we found that the rs422668 C allele may disrupt the binding effect of CXADR and mi R-1276.Subsequently,the dual luciferase reporter gene experiment verified that the C allele of rs422668 may increase the expression of CXADR gene m RNA by decreasing the binding ability of mi R-1276 and CXADR in 293T cells.5.In the TCGA database,the expression level of CXADR m RNA in HBV-associated HCC cancer tissues was reduced compared with normal tissues adjacent to cancer（P=0.018）.RT-PCR was used to detect the expression of CXADR in 78 pairs of HCC tissue and adjacent tissue samples,the results showed that CXADR m RNA was also expressed at a low levels in liver cancer tissues（P=0.0014）,and the low expression of CXADR was associated with an increased risk of death in patients with HBV-associated HCC after surgery（P=0.0348）.In the c Bioportal database,we found that the somatic mutation rate of the CXADR gene in HCC cancer tissues is low,suggesting that the change in its expression level may be mainly caused by SNP.In the TIMER 2.0 database,we found that the expression level of CXADR m RNA was positively correlated with the infiltration level of CD4+T cells,CD8+T cells and regulatory T（Tregs）cells in the tumor microenvironment（P<0.05）.6.In the GTEx database,we found that the COLEC12 rs8085897 G allele is correlated with the decrease in the expression level of COLEC12 m RNA in 760whole blood cells of healthy population(P=1.3×10^-4).After analyzing the somatic mutation of COLEC12 gene in liver cancer tissues,it was found that the gene has a certain degree of somatic mutation rate（HCC（Inserm）1.65%,HCC（AMC）0.87%,HCC（TCGA Pan Can）0.27%）,suggesting that the somatic mutation may plays a important role in affecting COLEC12 gene expression level.In the TCGA database,COLEC12 m RNA is highly expressed in liver cancer tissues and was associated with a poor prognosis of patients.Our study also found that the expression level of COLEC12 m RNA was positively correlated with the level of CD4+T infiltration,and the level of infiltration of CD8+T cells and Tregs cells.However,our study did not find that SIGLEC6gene is differentially expressed in HCC cancer tissues and adjacent tissues and is related to the prognosis of patients.Ⅱ Associations between SNPs of the complement system genes and OS of HBV-associated HCC after hepatectomy1.Through the association analysis of the discovery set and the validation set,we found that 8 SNPs（C6（complement component 6）rs4245976,C6rs140012002,C6 rs11951782,C6 rs10076104,C6 rs4245976,C6 rs6451568,TFPI（tissue factor pathway inhibitor）rs16829086 and TFPI rs3217130）were significantly associated with the postoperative OS of HBV-associated HCC patients.Then,through functional annotation,linkage disequilibrium analysis and multivariate stepwise regression analysis,we found that C6 rs4245976 C>T,TFPI rs3217130 C>T,TFPI rs16829086 AAAC>-（the mutation site is AAAC 4base deletion）were still independently associated with postoperative OS in HBV-associated HCC patients（all P<0.05）.Under the dominant model analysis of the overall dataset,the death risk of patients with C6 rs4245976 CT or TT genotype is 1.33 fold than that of patients with CC genotype（HR=1.33,95%CI=1.09-1.61,P=0.004）;The risk of death in patients with TFPI rs16829086 CT or TT genotype was 1.38 fold than that of patients with CC genotype（HR=1.38,95%CI=1.13-1.67,P=0.001）;while,the risk of death in patients with TFPI rs3217130 AAAC/-or-/-genotype was 0.71 fold than that of patients with CC genotype（HR=0.71,95%CI=0.57-0.87,P<0.001）.2.When combined the genotypes of these 3 significant SNPs（C6 rs4245976CT or TT,TFPI rs16829086 CT or TT and TFPI rs3217130 AAAC/AAAC）,we found that the more risk genotypes HBV-associated HCC patients carry,the worse the prognosis of patients had,it showed a dose-response effect(P_trend<0.001).Compared with HCC patients with 0 or 1 risk genotype,patients with 2or 3 risk genotypes had a 1.61-fold increase in postoperative mortality risk（HR=1.61,95%CI=1.32-1.96,P<0.001）.The results of ROC curve analysis show that compared with the prediction model constructed using clinical factors alone,the predictive model constructed based on the combined risk genotypes and various clinical factors can improve the ability to predict the 3 year or 5 year OS of patients with HBV associated HCC（3-year survival:72.72%vs.74.43%,P=0.023;3-year survival:72.04%vs.74.25%,P=0.011,respectively）.3.In the GTEx database,e QTL analysis results showed that the CC genotype of rs4245976 was associated with the decreased expression of C6 m RNA in 208liver tissues（P=0.031）.However,this study did not find that other SNPs genotypes were associated with the corresponding gene m RNA.4.Using the UALCAN database and Kaplan-Meier Plotter database,the results showed that the expression level of C6 m RNA in liver cancer tissues was significantly lower than that of adjacent tissues,and the difference was statistically significant(P<1×10^-12).We also found that the prognosis of liver cancer patients with low C6 expression was significantly worse（P=0.0071）.The expression level of TFPI gene m RNA in liver cancer tissues was lower than that in adjacent tissues,but it was not associated with the prognosis of liver cancer patients.We found that the somatic mutation rate of C6 gene and TFPI gene in HCC cancer tissues is low,suggesting that the changes in their expression levels may be affected by many different factors.5.We found that the expression level of C6 gene was negatively correlated with the infiltration level of CD4+T cells,CD8+T cells and Tregs cells in the tumor microenvironment using the TIMER 2.0 database（all P<0.05）.The expression level of TFPI gene was positively correlated with the infiltration level of CD4+T cells and Tregs in the tumor microenvironment(P=3.66×10^-2and 2.62×10^-3,respectively),and negatively correlated with the level of CD8+T cell infiltration(P=3.62×10^-3).Conclusion:1.These research results show that three SNPs of immunoregulatory genes CXADR rs2824339 C>T,COLEC12 rs8085897 C>G and SIGLEC6rs10221508 C>T were associated with postoperative OS of HBV-associated HCC patients after hepatectomy,and the combination of risk genotypes of these three SNPs（COLEC12 rs8085897 CC,CXADR rs2824339 CC and SIGLEC6rs10221508 CT or TT）synergically increased the risk of postoperative death in patients.2.Complement system-related genes SNPs（C6 rs4245976 C>T,TFPI rs3217130 C>T,and TFPI rs16829086 AAAC>-）were significantly correlated with postoperative OS of HBV-associated HCC patients after hepatectomy.With the increase of the number of risk genotypes（C6 rs4245976 CT or TT,TFPI rs16829086 CT or TT and TFPI rs3217130 AAAC/AAAC）,the risk of postoperative death of HCC patients increased in a quantitative way,that indicated that postoperative OS of patients with multi-risk genotypes was shortened and the prognosis was poor.3.The SNP rs422668 C allele may promote the expression of CXADR m RNA by decreasing the binding ability of mi R-1276 and CXADR,which may affect the prognosis of patients.4.COLEC12 m RNA is highly expressed in HCC cancer tissues,which is positively correlated with the infiltration level of a variety of tumor immune cells,and the prognosis of HCC patients with high COLEC12 expression is significantly worse.5.The low expression of C6 in liver cancer tissues was found,and was related to the infiltration of immune cells in the tumor microenvironment.the prognosis of liver cancer patients with low expression of C6 was significantly worse.