| Objective:Hepatocellular carcinoma (HCC) was the third common cause of cancer mortality all round the world. Remarkably, China alone accounts for about50%of all HCC cases worldwide. Due to the coherent distribution of HBV infection with HCC in China, HBV chronic infection is prevalent and seems to be particularly for importance. However, only a part of chronic carriers of HBV develop HCC,indicating that host genetic factors may also be involved in development of HBV-related HCC.This has also been proved by several candidate gene and genome-wide association study (GWAS) on HBVrelated HCC in eastern Asians.It is well known that DNA repair plays an essential role in genomic maintenance to prevent malignant transformation of normal cells. In eukaryote, one of the most lethal forms of DNA damage is the DNA double-strand break (DSB), as both strands of the DNA duplex are impaired simultaneously. The major repair pathways that repair DSBs are nonhomologous end-joining (NHEJ) and homologous recombination (HR). As a key member involved in the HR pathway, human RAD52exists in an oligomeric form, binds single-stranded DNA(ssDNA), promotes ssDNA annealing, and simulates RAD51-mediated homologous DNA pairing under certain specialized conditions. In human cells with the deficient RAD52, PALB2, or BRCA2gene,RAD52depletion could decrease clonogenic survival through increasing damage-induced chromosomal abnormalities and reducing the HR rates. Therefore, RAD52appears to exist in a synthetic lethality relationship with the RAD52,PALB2, or BRCA2gene. All these evidences demonstrated that RAD52might be important to preserve genomic integrity and prevent cancer development.As an important member in homologous recombination repair, RAD52plays a crucial part in maintaining genomic stability and prevent carcinogenesis. Methods:Several cancer susceptibility RAD52single nucleotide polymorphisms (SNPs) have been identified previously. For instance, there is an rs7963551A>C polymorphismin the3’-untranslated region (3’-UTR) of RAD52, which could reduce the binding affinity of miRNA let-7and, thus, elevated the transcription of the RAD52gene. In HCC cells, the deregulation of the expression of the let-7family of miRNAs by HBx may represent a potential novel pathway through which HBx acts to deregulate cell proliferation leading to hepatocarcinogenesis. It has been found that rs7963551and other SNPs in RAD52may be associated with the risk of multiple cancers, including breast cancer, lung cancer, thyroid cancer, head and neck cancers, as well as ovarian cancer. Therefore, we investigated the association between fiveRAD52SNPs (rs1051669, rs10774474, rs11571378, rs7963551, and rs6489769) and HBV-related HCC risk as well as its biological function in vivo. Considering the importance of RAD52in malignant transformation, we conducted two large independent case-control studies to investigate the association between RAD52genetic polymorphisms and risk for developing HBV-related HCC. To validate the biological function of RAD52SNPs in vivo, we examined the association between RAD52genotypes and mRNA expression levels in normal liver tissues.This study consisted of two case-control sets:(a)Shandong set (Discovery set):1186patients with HBV-related HCC, sex-and age-matched (±5years)508chronic HBV carriers as well as sex-and age-matched1308healthy controls. HBV-related HCC patients and chronic HBV carriers were recruited between June2009and October2012at Shandong Cancer Hospital, Shandong Academy of Medical Sciences (Jinan, Shandong Province, China). Control subjects were randomly selected from a pool of4500individuals from a community cancer-screening program for early detection of cancer conducted in Jinan city during the same time period as the patients were collected.(b) Jiangsu set (Validation set):560HBV-related HCC patients from Huaian No.2Hospital (Huaian, Jiangsu Province, China) and sex-and age-matched566controls. Patients were consecutively recruited between January2009and September2012at Huaian No.2Hospital. Controls were cancer-free individuals selected from a community cancer-screening program (3000individuals) for early detection of cancer conducted in Huaian city during the same time period as the patients were collected. The diagnosis of all patients was confirmed by a pathological examination combined with positive imaging (magnetic resonance imaging and/or computerized tomography). All participants were negative for antibodies to hepatitis C virus,hepatitis D virus or HIV since we excluded all participants with hepatitis C virus, hepatitis D virus or HIV infection. Chronic HBV carriers and HBVrelated HCC cases were defined as individuals with the following serological parameters [HBsAg(+) for>6months, anti-HBc(+), and anti-HBs(-)]. Healthy controls are ones without HBV infection. Individuals who smoked one cigarette per day for over1year were considered as smokers. Subjects were considered as alcohol drinkers, if they drank at least once per week.Forty-four normal liver tissues were obtained from surgically removed specimens of HCC patients in Huaian No.2Hospital and qianfoshan Hospital (Jinan, Shandong Providence, China).All RAD52SNPs (rs1051669, rs10774474, rs11571378, rs7963551, and rs6489769) were analyzed by the MassArray system (Sequenom,Inc, San Diego, CA). A15%blind, random sample of study subjects was genotyped in duplicates and the reproducibility was100%.Real-Time Analyses of RAD52mRNA SYBR-Green real-time quantity PCRmethod was used to examine RAD52mRNA levels in normal liver tissues as described previously. In brief, total RNA was isolated and converted to cDNA using the ReverTra Ace qPCR RT kit (TOYOBO,Osaka, Japan). Relative gene expression quantitation for RAD52and β-actin as an internal reference gene was carried out using the ABI7500real-time PCR system in triplicates. The primers used for RAD52were5’-CTGCGCGGCCTCCATCTAA-3’ and5’-GATTCTGGTTGACCTGCGCC-3’; and for bactin were5’-GGCGGCACCACCATGTACCCT-3’ and5’-AGGGGCCGGACTCGTCATACT-3’.The expression of individual RAD52measurements was calculated relative to expression of β-actin using the method as described previously.Results:In terms of median age and sex distribution, there was no statistically significant differences between HBV-related HCC patients, chronic HBV carriers and healthy controls for Shandong set and Jiangsu set (all P>0.05), indicating that the frequency matching was appropriate (Table1). Firstly, unconditional logistic regression analysis was used to examine associations between five RAD52SNPs(rs1051669, rs10774474, rs11571378, rs7963551, and rs6489769) and HBV-related HCC risk in Shandong discovery set (Table2). All observed genotype frequencies in both controls and patients conform to Hardy-Weinberg equilibrium in Shandong set consisting of1186patients with HBV-related HCC,508chronic HBV carriers as well as1308healthy controls. Logistic regression analyses revealed that rs7963551SNP was significantly associated with HBV-related HCC risk (Allelic OR=0.81,95%CI=0.67-0.99, P=0.031,HCC cases vs. chronic HBV carriers; allelic OR=0.80,95%CI=0.69-0.93, P=0.003, HCC cases vs. healthy controls)(Table2). However, the remaining four RAD52SNPs (rs1051669, rs10774474,rs11571378, and rs6489769) seem to be not risk variants for HBV-related HCC in Hari Chinese (all P>0.05)(Table2). The rs7963551C allele was showed to be protective allele;individuals with the CC genotype had an OR of0.67(95%CI=0.51-0.89, P=0.005, HCC cases vs. chronic HBV carriers) or0.69(95%CI=0.55-0.87, P=0.002,HCC cases vs. healthy controls) for developing HBV-related HCC, respectively, compared with individuals with the AA genotype (Table3). No such association between rs7963551and chronic HBV infection was observed (P>0.05)(Table3). Thus, we believe that the positive association between the rs7963551genotypes and HBV-related HCC was not due to predisposition to HBV infection. The association of HBV-related HCC risk with the rs7963551SNP was further validated in an independent case-control set. Genotyping results showed that this RAD52SNP was significantly associated with HBV-related HCC risk in Jiangsu Chinese population (Table3). Carriers of the rs7963551CC genotype showed significantly and consistently decreased risk to develop HBV-related HCC compared with rs7963551AA carriers (OR=0.41,95%CI=0.22-0.79, P=0.008)(Table3). In the pooled analyses, we found that the odds of having the rs7963551CC genotype in patients was0.59(95%CI=0.45-0.78,P=1.5×10-4,HCC cases vs. chronic HBV carriers) or0.65(95%CI=0.52-0.81, P=1.1×10-4,HCC cases vs.healthy controls) compared with the AA genotype (Table3). Since rs7963551A-to-C change could lead to decreased affinity between let-7and RAD52mRNA and increased RAD52expression in cancer cells,we examined whether there is an allele-specific effect of rs7963551SNP on RAD52expression in44normal liver tissues. As shown in Figure1, we found that subjects with the rs7963551AA genotype had significantly lower RAD52mRNA levels than those with the AC or CC genotypes in normal liver tissues.Conclusions:We found that only the RAD52rs7963551SNP was significantly associated with HCC risk, with the odds of having the rs7963551CC genotype in patients was0.59(95%CI=0.45-0.78, P=1.5×10-4, HCC cases versus chronic HBV carriers) or0.65(95%CI=0.52-0.81,P=1.1×10-4, HCC cases versus healthy controls) compared with the AA genotype. In the genotype-phenotype correlation analyses of44human liver tissue samples, rs7963551CC or AC was associated with a statistically significant increase of RAD52mRNA expression, which are consistent to functional relevance of allelic regulation of RAD52expression by rs7963551SNP and miRNA let-7in cancer cells.Significance:Our data demonstrated that RAD52functional rs7963551SNP contributes to susceptibility to developing HCC. |
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