Identification Of Sequence Polymorphisms In The COX Region Of Mitochondrial DNA As A Risk Factor For Hepatocellular Carcinoma

Objective:Hepatocellular carcinoma is one of the most frequent cancer in the world and it is the second fatal tumor in China. It threatens nation health and economy due to the high mortality rate. Recently, it was demonstrated that the mitochondrial DNA(mtDNA) mutation and single nucleotide polymorphisms(SNPs) were associated with the occurrence of cancer. The current studies were limited to mtDNA non-coding regions, but few involved in mtDNA coding regions. COX gene is located in mtDNA coding region and responsible for coding three subunits of cytochrome oxidase(COX). COX plays an important role in oxidative phosphorylation(OXPHOS) process and carcinogenic mechanism, and its mutant and polymorphism may be associated with tumor. SNPs is the polymorphisms induced by single nucleotide variation in the DNA sequence, which has been widely applied to research susceptible genes of multigenic disease. In this study, PCR amplification and sequencing the entire section of COX gene were finished. It is proposed to find the SNPs relating with the cancer risk in hepatocellular carcinoma(HCC) patients using case-control study. The relationship between the HCC risk-associated SNPs and clinical features was evaluated, and the potential role of mtDNA polymorphisms in occurrence and progression of tumors was explored, which would be helpful to detect valuable markers for HCC risk evaluation and make therapeutic decisions earlier.Methods:1 Samples: Tissue samples were collected from 130 HCC patients at Fourth Hospital of Hebei Medical University, 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 9 paired primers were designed according to NCBI database(http://www.ncbi.nlm.nih.gov/ snp/). 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 Statistical analysis: The entire mitochondrial COX gene was checked for meaningful SNPs, then enlarged the sample size of these valuable sites for further study.The student’s t-test and u-test were used to analyse differences of clinical features between HCC patients and age-matched controls. The χ2 test was used to analyse the genotype frequencies of SNPs. All analyses were performed using SPSS software(version 21.0). A P-value <0.05 was considered statistically significant, and all statistical tests were two sides.Results:1 As for clinical characteristics, there was no statistical difference in age and gender between HCC group and heathy controls(P>0.05).2 The SNPs analysis of the 9 fragments were performed in 30 HCC patients and number-matched controls. 6962G/A、7196C/A、7853G/A、8701A/G、8584G/A、8414C/T、9180A/G、9540T/C、9545 A/G were chosen due to their high frequency(>5%). But only the SNP 9545 A/G in COIII was found in the further statistical analysis, the frequency distribution was critical difference between HCC patients and healthy controls(χ2=5.455, P=0.052). As a consequence, the SNP 9545 A/G was chosen for further study.3 After expanded the genetic sample size of 9545 A/G in COIII and for further statistical analysis, For the SNP 9545A/G targeting in COIII, the genotype distribution frequency between A/A and G/G was different at signicant levels(χ2=5.559, P=0.026). It was demonstrated the SNP 9545A/G was associated with the cancer risk for hepatocellular carcinoma development in the data.4 The relationship between the HCC risk-associated SNPs and clinical features of HCC was then analyzed. The result showed: there was no association of 9545A/G SNPs with clinical features, including gender、age、TNM classification 、size of the tumor、portal vein thrombosis and Child classification.Conclusions:1 For the SNP 9545 A/G targeting in COIII, the frequency distribution was significant difference between HCC patients and healthy controls. The SNPs for 9545 A/G was associated with the cancer risk for hepatocellular carcinoma development.2 No association exist for the genotype distribution of 9545 A/G with clinical characteristics( gender、age、TNM classification 、size of the tumor、portal vein thrombosis and Child-Pugh classification) in HCC(P>0.05).