| Objective:Hepatocellular carcinoma(HCC)is the third leading cause of cancer mortality in China and there are an increasing number of cases in the recent decades.Most of the patients with HCC are diagnosed at the late stage,for which the 5-year survival rate is less than 10%.At present,the treatment of hepatocellular carcinoma is mainly represented by surgery,chemotherapy and radiotherapy,but the overall prognosis is poor.Therefore,it is necessary to find a more effective adjuvant therapy to improve the therapeutic effect of hepatocellular carcinoma.About 80%of HCC patients in China have a history of hepatitis B virus(HBV)infection,which reflects the relationship between chronic infection and the prevalence of HCC.The development from viral hepatitis and cirrhosis to hepatocellular carcinoma is an important process of hepatocarcinogenesis.However,only a few chronic HBV carriers develop hepatocellular carcinoma,suggesting that host genetic factors may also be involved in the pathogenesis of hepatocellular carcinoma.After DNA injury,the absence of cell cycle checkpoint can be used as a marker for the occurrence of hepatocellular carcinoma.Some studies have shown that DNA double strand breakage can significantly improve the integration rate of HBV,thus promoting the occurrence and development of HCC.As an important DNA double strand repair related gene,NBS1 gene plays an important role in maintaining genomic stability and preventing cell carcinogenesis.It also plays an important role in maintaining telomere stability,opening immunoglobulin and meiotic recombination,and promoting embryonic development.Epidemiological data shows that NBS1-deleted heterozygotes may not appear to be associated with clinical symptoms,but the risk of malignant tumors is still significantly increased in these patients,especially breast cancer,prostate cancer,rectal cancer,leukemia and non-Hodgkin's lymphoma,but there is no clear evidence of its association with liver cancer.NBS1 gene mutation can promote the occurrence of liver cancer in mice,multiple single-nucleotide polymorphism(SNP)sites can increase the risk of some cancers.So far,there is no large case report of NBS1 SNP in HBV related HCC,and there are few studies on the mechanism of gene regulation.On the basis of previous reports,the correlation between NBS1 gene polymorphism and hepatocellular carcinoma(HCC)caused by chronic HBV infection was analyzed,and then the relationship between rs1805794C/G polymorphism and susceptibility to various cancers was further clarified by Meta-analysis.Finally,the mechanism of gene regulation on cell.function was explored.Methods:508 patients with hepatocellular carcinoma(HCC)associated with HBV infection were randomly selected as the experimental group,481 patients with chronic hepatitis B infection with matched personality and age,and 581 healthy people without HBV infection as the control group,all the individuals were recruited at the Affiliated Hospital of Shandong Academy of Medical Sciences.Several SNP loci were selected from previous genome-wide association studies(GWAS)and NBS1 candidate gene association studies.Genomic DNA was extracted from whole blood samples and genotyping was performed by Taqman.The difference of age,sex,smoking and drinking status between the patients and the control group was evaluated by bilateral chi-square test(P<0.05).The genotype phenotype relationship of each group was analyzed by Plink software and Cochran-Armitage trend test.Bonferroni calibration was performed on multiple SNP loci,and P<0.0125 was considered to be significantly correlated.In order to further clarify the relationship between the rs1805794C/G polymorphism and cancer susceptibility,we used Meta-analysis to make a systematic review of relevant studies.We retrieved clinical studies published in Pubmed and Embase from 2008 to 2008 on NBS1 rs 1805794 associated with cancer susceptibility and evaluated the quality of the included literature.The association between NBS1 rs1805794C/G polymorphism and cancer was evaluated by odds ratio(OR)and 95%confidence interval(95%CI).Subgroups were classified according to race,source of control group and type of cancer to evaluate the impact of these factors on Association analysis.Finally,sensitivity and publication bias were assessed.HepG2 cells were treated with different concentrations of NBS1-siRNA for different time and the expression of NBS1 gene was detected.CCK-8 assay and flow cytometry were used to analyze the proliferation and apoptosis of HepG2 cells before and after transfection.Finally,the cell cycle was detected by PI staining and flow cytometry.HepG2 cells were transfected with.medium concentration of siRNA to inhibit the expression of NBS1 Total intracellular mRNA was extracted 24 hours or 48 hours later.FluiDigm high-throughput gene analyzer was used to quantify 48 specific genes.Beta-actin and GAPDH were internal reference genes.Differential expression genes(DEGs)were screened according to | Fold Change |>2.0,P<0.05.Gene ontology(GO)was used to analyze the differentially expressed genes at the above two time points(P<0.05).The smaller the P value,the more meaningful the GO Term is.The GO Term ranked in the top 10 was selected for analysis.The differentially expressed genes were enriched by Kyoto Encyclopedia of genes and genomes(KEGG)signal pathway and the signal pathway was selected according to the P<0.05 standard.Results:We selected five NBS1 SNPs that may cause liver cancer,namely rs10464867,rs1063053,rs1061302,rs1805794,and rs709816.The participants' whole blood DNA samples were analyzed and four loci(rs10464867,rs1063053,rs1805794,rs709816)were successfully typed.We found that these four SNPs were not significantly associated with HCC or chronic hepatitis B and HBV infection.It is noteworthy that we found that NBS1 rs1805794 plays an important role in the progression of HCC in patients with chronic hepatitis B(P=2.99E-03,OR=1.31).After a series of screening,21 papers(including 25 independent studies)were included in our Meta-analysis,including 10901 cancer cases and 14124 controls.The genotypes of the control groups were consistent with the H-W balance and the literature quality scores were 6.5-8.5.Overall,carrying the C allele increased the risk of cancer(C vs.G:OR=1.21,95%CI:1.08-1.36,P = 0.001).Subgroup analyses revealed that,carrying C alleles increased the risk of liver cancer(OR=1.37,95%CI:1.13-1.67,P=0.001),leukemia(OR=1.77,95%CI:1.19-2.63,P=0.005)and nasopharyngeal carcinoma(OR=1.98,95%CI:1.76-2.24,P<0.001).NBS1 rs1805794C/G polymorphism in all genetic models of nasopharyngeal cancer,most genetic models of liver cancer and leukemia was significantly associated with increased risk of cancer.There was a significant association between the rs1805794C/G polymorphism and the increased risk of cancer in the Asian population,but no significant statistical significance was found in the Caucasian population.There was a significant association between rs1805794C/G and increased cancer risk in the population-based(PB)studies,but no significant statistical significance was found in the studies of which control groups based on hospitial(BP).All the genetic models showed significant heterogeneity(P<0.001),so the random effect model was used.After subgroup analysis,the.heterogeneity of breast cancer,colorectal cancer,liver cancer,lymphoma,nasopharyngeal cancer,Caucasian population and population-based control group was significantly reduced,and P>0.1 was found in most of the gene models.No statistically different ORs were observed when excluding every single study by sequence,so the results verified the stability of our meta-analysis.The Begg's funnel plot showed that the distribution of the included studies was symmetrical and inverted,indicating no significant publication bias.Compared with the control group,the viability of NBS1-siRNA transfected cells was significantly decreased(P<0.05 or P<0.01).After 24 hours of transfection,the cell proliferation rate decreased slightly,but there was no significant difference(P>0.05);with the increase of transfection time,the cell proliferation rate decreased sharply.When the transfection time was 48 hours,the cell proliferation rate decreasedmost significantly;after 72 hours of transfection,the cell proliferation capacity was still higher than that of non-transfected cells.But the transfection rate was not significantly different after 48 hours.Overall,the cell proliferation rate was the lowest when 50 nM siRNA was transfected into cells,and the effect was the most significant after 48 hours.Flow cytometry was used to observe the apoptosis of siRNA after 48 hours of transfection.Compared with the untransfected cells,the apoptosis rate increased significantly after transfection(P<0.05 or P<0.01),and it was more significant with the increase of concentration.After interfering with NBS1 expression with medium and high concentration of siRNA,the proportion of HepG2 cells in S phase increased(P<0.05).In combination with the above results,we hypothesized that interfering with NBS1 expression could inhibit cell proliferation by blocking cells at S phase.Compared with the control group,14 genes were up-regulated and 7 weredown-regulated after 24 hours of transfection.After 48 hours of transfection,15 genes were up-regulated and 5 were down-regulated.The clustering map showed that there were 11 differentially expressed genes at the two time points,of which 8 genes were up-regulated,including IRS1,MET,CCND1,IFG2,PTK2,YAP1,MYC and PTEN,and 3 genes were down-regulated,including CDH13,TGFB1 and AKT1 respectively.These genes may serve as a downstream target for NBS1 to regulate the progression of hepatocellular carcinoma.GO analysis showed that the differentially expressed genes interfered with NBS1 not only affected the basic function of cell life,but also regulated cell cycle and cell migration.KEGG results showed that the differentially expressed genes were mainly involved in the signal pathways including cancer pathway,prostate cancer and colorectal cancer.Conclusion:1.The rs1805794 C/G polymorphism of the NBS1 gene increases the risk of hepatocellular carcinoma in HBV-infected individuals,which may be a genetic predisposing factor for HCC in chronic HBV-infected Chinese Han population.2.Through Meta-analysis,we know that rs1805794 C/G polymorphism may play a crucial role in increasing the susceptibility to cancer,especially in liver cancer,leukemia,nasopharyngeal carcinoma and Asian populations.3.Interference with the expression of NBS1 in HepG2 cells can arrest cell cycle at S phase and inhibit cell proliferation and induce cell apoptosis.Through further experiments and bioinformatics analysis,we have a preliminary understanding of the downstream regulation of NBS1 target genes and pathways.Significance:1.We revealed the relationship between NBS1 SNPs and HBV related HCC susceptibility in Chinese Han population.2.We further reviewed the relationship between NBS1 rs1805794 C/G polymorphism and susceptibility to various cancers including liver cancer in different ethnic groups.3.We demonstrated the effect of NBS1 gene on the biological function of HepG2 and initially explored the mechanism of gene regulation on the function of hepatocellular carcinoma cells.4.The study will be helpful to reveal the pathogenesis of HCC from the perspective of gene mutation and signal pathway,and provide theoretical basis for early diagnosis and targeted therapy of HCC with NBS1 mutation and abnormal expression. |
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