Genetic Polymorphisms In MicroRNA-related Genes Modify The Susceptibility Of Hepatocellular Carcinoma In Chinese Han Populations

Primary liver cancer is one of the most prevalent malignancies in the world.Hepatocellular carcinoma (HCC) represents the major histological subtype,accounting for70%to85%of the total liver cancer burden worldwide. Liver cancerin men is the fifth most frequently diagnosed cancer worldwide but the second mostfrequent cause of cancer death. In women, it is the seventh most commonly diagnosedcancer and the sixth leading cause of cancer death. An estimated748,300new livercancer cases and695,900cancer deaths occurred worldwide in2008. The incidencerate of HCC varies considerably worldwide, with the highest incidence rates in Eastand Southeast Asia and Sub-Saharan Africa, and China alone accounted forapproximately55%of all HCC malignancies. Major etiologic factors of HCC includeinfection with hepatitis B virus (HBV) and hepatitis C virus (HCV), Aflatoxin B1exposure and possibly cigarette smoking and alcohol drinking. Overall,75–85%ofHCC patients are attributable to persistent viral infection of HBV, especially indeveloping countries. Overall,75%–80%of cases of HCC are attributable topersistent viral infections with either HBV (50%–55%) or HCV (25%–30%). It isestimated that2billion people have been infected with HBV, with an estimated300million persons with chronic infection worldwide. However, it also is known thatonly a minority of lifelong chronic carriers of HBV eventually will develop HCC, thus implicating the importance of cofactors (environmental or genetic) inHBV-related HCC. Genetic determinants also play an important role in the etiologyof HCC, including variants located at both coding and noncoding sequences.MicroRNAs (miRNAs) are non-coding, single-stranded RNAs of~22nucleotides and constitute a novel class of gene regulators through binding to the3’-untranslated region (UTR) of protein-coding transcripts, in turn triggeringmessenger RNA degradation or translational repression. MiRNAs are initiallyproduced as large RNA precursors that are called pri-miRNAs through RNApolymerases II. Pri-miRNAs are cleaved to precursor miRNAs (pre-miRNAs) of~70-nucleotide by Drosha (RNAse III). Then, XPO5in a complex with RANrecognize and bind to the pre-miRNA molecule, exporting it to the cytoplasm. It isfurther processed to double-stranded duplexes of20-23nucleotides (mature miRNAs)by a protein complex including DICER, AGO1and AGO2. A number of studies havehighlighted the role of miRNA in various biological processes, even the developmentof cancers. Recent studies revealed a subset of miRNAs dysregulated in HCC, andcould be used as biomarkers for early detection and prognosis prediction. Because ofthe functional relevance of miRNAs, we hypothesized that potentially functionalpolymorphisms in3’-UTR of miRNA processing genes and primary miRNA mayinfluence the miRNA maturation and then modify HBV infection and HCC risk.To test these hypotheses, case-control study was performed to have thefollowing aim:(1) To explore the relationship between the functional geneticpolymorphisms on miRNA biosynthesis pathway and HCC susceptibility of ChineseHan people.(2) To evaluate the correlation between the hereditary variants in primarymiRNA or precursor miRNA and risk of HCC in the Chinese Han population. Theresults of this study will be helpful to determine the genetic factors of HCC inChinese population and elucidating the susceptibility mechanism in HCC, andprovide a scientific basis for high-risk population screening and early diagnosis andtreatment of HCC. PartⅠ Potentially Functional Genetic variants in microRNAprocessing genes and risk of HBV-relatedhepatocellular carcinomaGenetic variations in miRNA processing genes may affect the biogenesis ofmiRNA, hence risk of HBV infection and hepatocellular carcinoma (HCC)development. In the present study, we hypothesized that potentially functionalpolymorphisms in3’-untranslated region (UTR) of miRNA processing genes may beregulated in turn by miRNA and then contribute to susceptibility of HBV infectionand HCC development. To test the hypothesis, we genotyped three selected SNPs(rs1057035in DICER1, rs3803012in RAN and rs10773771in PIWIL1) in acase-control study of1300HBV-positive HCC cancer cases,1344HBV persistentcarriers and1344HBV natural clearance subjects in Chinese. We observed thatDICER1rs1057035CT/CC variant genotypes were associated with a significantdecreased risk of HCC (adjusted OR=0.79,95%CI=0.64-0.96) compared withwild-type TT and RAN rs3803012AG/GG variant genotypes increased the risk ofHBV persistent infection compared with AA genotype (adjusted OR=1.35,95%CI=1.03-1.77). However, PIWIL1rs10773771CT/CC variant genotypes wereassociated with an approaching decreased risk of HCC (adjusted OR=0.86,95%CI=0.73-1.01) and similar with RAN rs3803012AG/GG (adjusted OR=0.80,95%CI=0.61-1.06). In addition, the combined effects of these three SNPs showed alocus-dosage effect on HCC risk (Ptrend=0.001). Furthermore, reporter gene assaysindicated that the three SNPs (rs1057035, rs3803012and rs10773771) might changethe binding ability of miRNAs to the3’UTR of the three genes (DICER1, RAN andPIWIL1), respectively. These findings indicated that DICER1rs1057035, RANrs3803012and PIWIL1rs10773771might contribute to the risk of HBV-relatedHCC. Part II A Genetic Variant in Primary miR-378and Risk ofHepatocellular CarcinomaMiR-378has been reported to be related to cell survival, tumor growth andangiogenesis and may participate in hepatocellular carcinoma (HCC) development.Genetic variants may impact miR-378expression and its function. In this study, wehypothesized that single nucleotide polymorphism (SNP) rs1076064in primarymiR-378(pri-miR-378) may contribute to HCC susceptibility through alteringmiR-378expression. We conducted a case-control study and genotyped rs1076064in1300hepatitis B virus (HBV) positive HCC patients,1344HBV persistent carriersand1344subjects with HBV natural clearance. Reporter gene assay was conducted toevaluate the functional relevance of the SNP rs1076064. We found that the variantgenotypes of rs1076064were significantly associated with HCC risk in HBVpersistent carriers in an additive genetic model (adjusted OR=0.90,95%CI=0.81-1.00, P=0.047). No significant association was found between rs1076064andHBV clearance. The reporter gene assay supported that the variant G allele ofrs1076064exerted higher promoter activity than the A allele. Overall, these findingssuggested that the variant genotypes of rs1076064in pri-miR-378may contribute toHCC susceptibility in HBV persistent carriers through altering pri-miR-378transcription.