| Primary liver cancer is one of the most common malignancies worldwide, of which 85% to 90% are hepatocellular carcinoma (HCC). More than an half of the new cases and cancer deaths of HCC in China. Due to the malignancy and poor prognosis of HCC, most patients tend to have advanced to the time of diagnosis, and thus delay the best time of surgical treatment. Even with resection, recurrence rates of HCC patients can be as high as 80% for two years, of which the survival rate after five years is about 10% to 20%. And five-year survival rate of patients with advanced disease is less than 3%. Guangxi has high incidence of HCC, of which the HCC mortality ranks first in spectrum of tumor death, harming to people’s health and lives. Major etiologic factors of HCC include environmental exposure factors and individual genetic susceptibility. But so far, the most important cause, pathogenesis and prognostic factors of HCC is still not very clear. Numerous studies show that individual genetic variation has a significant impact on risk of the disease, clinical treatment and prognosis of the disease. Important aspect of single nucleotide polymorphisms (SNPs) is to determine the individual genetic variation, is currently the most widely used genetic markers. Currently, some research suggests that SNPs are associated with the development, clinical features and prognosis of malignant tumors. But association between miRNA gene polymorphisms and susceptibility to HCC and prognosis were rarely reported. microRNAs (miRNAs) are a class of endogenous single-stranded RNAs, and involves in regulating the expression of downstream target genes highly conserved. miRNAs constitute a novel class of gene regulators through binding to the 3’-untranslated region (UTR) of protein-coding transcripts, in turn triggering messenger RNA degradation or translational repression. miRNAs play a role in a variety of malignancies as oncogenes, up-regulated in tumors, and also as suppressor genes, down-regulated in tumors. miRNA-122 was a liver-specific high-expressed miRNA in human, which has been shown play a vital role in normal liver functions, including hepatocyte phenotype, differentiation, development, and metabolism, immune response and and other physiological processes, as well as in the pathological changes, including in chronic HBV infection and HCC. miRNA-199 is an important regulatory factor as tumor suppressor gene. Studies have shown that, miRNA-199 plays an important role in the occurrence and development of HCC. miRNA-199 was up-regulated in normal human liver, but down-regulated in HCC, which could reduce survival rate of HCC. Another study also found that, miRNA-199 might prevent the invasion and metastasis of tumor cells through negative regulation of the proto-oncogene c-Met and its downstream effectors ERK2.Recent studies show that, miRNA-related SNPs can affect the maturation process of miRNA and expression of target genes and the recognition and binding with target genes, so that abnormal miRNA regulatory networks, thus affecting tumor occurrence, development and prognosis. miRNA-related SNPs including SNPs of miRNA gene, SNPs of biosynthetic pathway genes and SNPs of miRNA target sequences. Because of the mature miRNAs molecules shorter, their SNPs likely to affect the maturation process of miRNA and downstream genes. And this assumption has been proved in a series of genome research and vitro experiments. Given the role of miRNA-122 and miRNA-199 in HCC process, therefore, miRNA-122 and miRNA-199 gene SNPs may affect regulatory networks, thus affecting the risk, clinical features and prognosis of HCC. This study was divided into two parts. In the first part, a case-control study was conducted to explore miRNA-122 and miRNA-199 gene SNPs and genetic susceptibility to HCC. In the second part, clinical data of HCC cases were collected and follow-up to investigate miRNA-122 and miRNA-199 gene SNPs and clinical features and prognosis of HCC.Part IThe association of single nucleotide polymorphism in miRNA-122 and miRNA-199 with susceptibility to Hepatocellular carcinomaObjectTo explore the association between SNPs in miRNA genes and susceptibility to HCC in Guangxi population. SNPs include miRNA-122 gene rs9966765, rs1135519, rs17669 and miRNA-199 gene rs74723057, rs12120556.And gene-gene, gene-environment interactions were also analyzed.Methods(1) miRNA-122 and miRNA-199 gene SNPs selectionIn accordance with a predetermined frequency standard, we use dbSNP database to select SNPs. Frequency standards are:minor allele frequency (MAF) of the candidate SNPs is 0.05 and each SNPs LD (r2) is less than 0.8. Finally, miRNA-122 genes rs9966765, rs1135519, rs17669 and miRNA-199 gene rs74723057, rs12120556 five SNPs were selected.(2) The research was designed as a case-control study based on hospital in a large sample. From June 2007 to June 2011,1050 newly histopathologically diagnosed HCC patients from First Affiliated Hospital of Guangxi Medical University and the First Affiliated Hospital of Guangxi Traditional Chinese Medicine University, first People’s Hospital of Liuzhou were enrolled as the case group. All the HCC cases never received chemotherapy and radiotherapy, with 1079 non-cancer patients in the Spinal Orthopedic Department-, Traumatic Department、Aesthetic Plastic Surgery and Ophthalmology as the control, with the case and the control matched in terms of ages, sex. The questionaire features include basic datum, disease history, family history, smoking history, and drinking history etc.2ml fasting venous blood were collected for genome DNA extracted. Finally five SNPs including miRNA-122 gene rs9966765,rs1135519、 rs 17669 and miRNA-199 gene rs74723057、rs12120556 were measured with Sequenom MassArray Genotyping System.(3) All data were input and analyzed statistically in SAS9.1.3 for Windows. Student’s t-test was used to compare mean value of the samples. While categorical data were compared with the use of the chi-square test. Unconditional logistic regression method was used to extract the OR value and its 95% confidence interval (95%CI) by adjusting for age, sex, smoking, alcohol consumption, and family history of cancer.Results(1) TT homozygous frequency distribution of miRNA-122 rs 1135519 in the cases and the control group was 68.65%,73.14%, respectively, TC heterozygous frequency distribution in both groups was 28.37%,25.09%, respectively, CC mutation homozygotes distribution of frequencies in the two groups were 2.98%, 1.77%, respectively, and frequency distribution of TT, TC, CC genotypes was significantly different in cases and the controls (P= 0.022). Adjusted for age, sex, smoking, alcohol consumption and HBV infection, CC genotype significantly increased the risk of HCC (OR= 2.708,95% CI:1.154-6.356). But we did not find SNPs in miRNA-122 gene rs9966765, rs 17669 and miRNA-199 gene rs74723057, rs12120556 loci were associated with HCC risk.(2) Stratified analysis showed that, miRNA-122 gene rs9966765, rs1135519 TT genotypes increase risk of HCC in older people (>the average age of 47) (adjustment OR=1.565,95%CI:1.063-2.303 for rs9966765; OR= 1.534,95% CI:1.043-2.257 for rs1135519). MiRNA-122 gene rs17669 AG/GG increase risk of HCC in older people (>the average age of 47) (OR= 1.482,95% CI: 1.003-2.192), and also increase HCC risk in women (OR= 2.240,95% CI: 1.035-4.484).(3) miRNA-122 rs17669 AG/GG genotypes and a history of alcohol consumption showed gene-environment interaction (P= 0.044). But the interaction between SNPs in miRNA-122 rs9966765, rs1135519 and miRNA-199 rs74723057, rs12120556 and a history of alcohol consumption were not found in the risk of HCC.(4) All SNPs in miRNA-122 gene rs9966765, rs1135519, rs17669 and miRNA-199 gene rs74723057, rs12120556 showed no Gene-gene interaction in the risk of HCC.ConclusionThe results of the study show that, miRNA-122 gene SNPs may influence genetic susceptibility to HCC in Guangxi region, and rs1135519 polymorphism may be an independent risk factor of HCC. The interaction between rs17669 SNP and a history of alcohol consumption increased HCC risk of populations in Guangxi.PartⅡThe association between miRNA-122>miRNA-199 gene polymorphism and prognosis of Hepatocellular carcinoma and its clinical characteristicsObjectiveTo evaluate association between SNPs in miRNA-122 rs9966765、rsl 135519、 rs17669、miRNA-199 rs74723057、rs12120556 and prognosis of HCC and its clinical characteristics, aimed at providing referenced evidences for the prognosis of HCC evaluation.MethodNew cases of 276 HCC patients who accepted radical resection or non-surgical treatment hospitalized at Guangxi Medical University First Affiliated Hospital and Guangxi Medical University Affiliated Cancer Hospital between February 2007 and February 2010 were included. We retrospectively reviewed the medical and pathology records for each patient, including the time of diagnosis, treatment, imaging findings, clinical features, and AFP levels, total bilirubin, and albumin and other laboratory results. This study included 179 patients who had undergone surgical resections for HCC at the Guangxi Medical University First Affiliated Hospital from 2008 to 2009, and follow-up information was available for every patient, including survival situation recurrence, metastasis and death situation, etc.. Sequenom MassArray was applied to detect the genotypes of candidate SNPs. All data were double entry and consistency checking by EpiData3.0 and analyzed statistically in SPSS 13.0 for Windows. The correlation between miRNA polymorphisms and clinicopathologic characteristics was analyzed using the χ2 test.Kaplan-Meier method was used to draw survival curve and univariate analysis, log-rank test was used to compare the difference of genotypes with overall survival (OS) and recurrence-free survival time (RFS), and the Cox proportional hazards model was used for multivariate analysis. The hazard ratios (HRs) and the associated 95% confidence intervals (CIs) were derived from a Cox proportional-hazards regression model.Result(1) Five SNPs genotypes of miRNA-122 rs9966765. rsl 135519、rs17669 and miRNA-199 rs74723057> rsl2120556 were not associated with clinical characteristics of HCC patients, including the general classification, HCC stage, tumor location, number of tumors, preoperative liver cirrhosis, portal vein thrombosis, vascular invasion, the envelope and the serum AFP, total bilirubin, albumin level (P> 0.05).(2) K-M Univariate analysis showed that, five SNPs of miRNA-122 and miRNA-199 were not associated with overall survival and recurrence-free survival of HCC patients statistically (P> 0.05).(3) Cox regression analysis adjusted for age, gender, smoking, drinking and HBV infection showed that, miRNA-122 rs1135519CC genotype was not associated with overall survival of HCC patients, but were protect factor of recurrence-free survival of HCC patients who had have surgical resections (adjusted HR=0.192,95%CI 0.043-0.858)。(4) Cox regression analysis adjusted for all the factors showed that, serum albumin, the number of tumors, vascular invasion, and miRNA-122 gene rs1135519 TC genotype were the risk of overall survival of HCC patients. AFP level, tumor type, envelope and miRNA-199 gene rs74723057 CG genotype were associated with recurrence-free survival of HCC patients who had have surgical resections.ConclusionmiRNA-122 gene rs1135519 polymorphism may affect the overall survival of HCC patients, and miRNA-199 gene rs74723057 polymorphism may be associated with recurrence-free survival of HCC patients who had have surgical resections. |
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