Single-nucleotide Polymorphisms In MicroRNA Binding-sites Are Potentially Associated With The Cancer Risk And Prognosis For Hepatocellular Carcinoma

Objective: MicroRNA(miRNA) is a small non-coding (~22 nt) RNA, which is encoded in the chromosomal DNA, and it can regulate gene expression at the post-transcriptional levels. Previous studies revealed that miRNAs play an important role in a broad range of biological processes, such as embryonic development, cellular proliferation and differentiation, apoptosis, and cancer development. MiRNA can lead to mRNA cleavage or translational repression so as to regulate targeted gene expression by pairing within 3'-terminal untranslated regions (3'UTR). Cancer risk associated with single nucleotide polymorphisms (SNPs) in miRNA binding site was identified by genotyping SNP in putative miRNA targeting region in recent studies. Some studies conducted a genome-wide analysis of miRNA targeting site SNPs and found 12 miRNA binding SNPs displaying an aberrant allele frequency in human cancers by using human cancer specimens (including various cancers as whole) against the dbSNP database for case-control study. In this study, we evulated 12 miRNA binding SNPs to identify the cancer risk and prognosis in hepatocellular carcinoma (HCC) patients.Methods: 1 Samples: Tissue samples were collected at Fourth Hospital of Hebei Medical University from 60 HCC patients, who underwent tumor resection in the Department of Hepatobiliary Surgery from September 2007 to March 2008. Normal blood samples were collected from age- and number-matched healthy controls.2 DNA extraction: Genomic DNA was extracted immediately with a Wizard Genomic DNA extraction kit (Promega) from tissue samples and normal blood samples. Then the DNA was frozen immediately in refrigerator until used.3 PCR amplification and sequence: The sequence of 12 paired primers were designed according to NCBI database (http://www.ncbi.nlm.nih.gov/ snp/), which can amplify DNA fragments flanking for the 12 cancer-associated SNPs in miRNA binding-sites. Polymerase chain reaction (PCR) was performed using a PCR Master Mix Kit according to manufactor's instructions(Promega), and purified before sequencing. Sequencing was done by Sangon Biotech (Shanghai) Company.4 Following up: we followed up one-year survival status of post- operational HCC patients by phones.5 Statistical analysis: The student's t-test was used to analyse differences of clinical features between HCC patients and age-matched controls. Theχ2 test was used to analyse the genotype frequencies of SNPs. The survival rates and survival curves were evaluated by the Kaplan-Meier method, and compared by the Log-Rank test.All analyses were performed using SPSS software (version 13.0, SPSS, Chicago, IL). A P-value <0.05 was considered statistically significant, and all statistical tests were two sides.Results: The SNPs analysis of the 12 miRNA binding sites were performed in 60 HCC patients and number-matched controls. Rs12583, rs1128665, rs17703261, rs14109, rs17107469 and rs10463, were elminated for further analysis due to unfitting Hardy-Weinberg equilibrium in 60 controls.1 The genotype distribution frequency for each SNP (rs16917496, rs1053667, rs11337 and rs3660) showed no statistical difference between HCC patients and matched controls. For the SNP rs1044129a targeting in RYR3, the genotype distribution frequency between A/A and A/G was different at signicant levels (χ2=5.562, P=0.018),and so it was for A/A versus A/G+G/G genotypes (χ2=4.261, P=0.039). As for the SNP rs4901706 targeting in C14orf101, the genotype distribution frequency between G/G and A/G was also significantly different(χ2=5.022, P=0.025), and so it was for G/G versus A/G+A/A genotypes (χ2=5.647, P=0.017) and G versus A allelotypes (χ2=4.843,P=0.028). The data demonstrated the SNPs rs1044129a and rs4901706 were associated with the cancer risk for hepatocellular carcinoma development.2 We compared the survival rates of post-operational HCC patients between different genotypes for each SNP in following study. No statistical difference was existed for genotype distribution of rs1044129a, rs4901706, rs16917496, rs11337 and rs3660. According to the genotypes T/T, C/T and C/C for rs1053667 targeting in KIAA0423, HCC patients were divided into 3 groups. The two-year survival rate for each genotype is 44.74%(T/T), 40.00%(C/T) and 0.00%(C/C) respectively. The rare allele genotype C/C seemed to be associated with the shortest survival length. We bloted the survival curves for these patients by Kaplan-Meier method at rs1053667. Compared with the T/T and T/C curves which displayed similar curved lines, the C/C curve droped remarkably. Significant difference for the survival rates between T/T and C/C genotype's patients was showed by Log-Rank test (P=0.0054). The rare allele C genotype seemed to shorten survival length with a recessive maner.Conclusions: 1 For the SNPs rs1044129a targeting in RYR3 and rs4901706 targeting in C14orf101, the frequency distribution for each SNP was significant difference between HCC patients and healthy controls. The SNPs for rs1044129a and rs4901706 were associated with the cancer risk for hepatocellular carcinoma development.2 For the SNP rs1053667 targeting in KIAA0423, there was significant difference in the survival rates of patients with T/T and C/C genotypes. The C/C genotype was associated with the shortest period of survival. The rare allele C genotype was negative correlation with survival rate of HCC patients. The rare allele C genotype seemed to shorten survival length with a recessive maner.