Mcm3ap, A Novel Hbv Integration Site In Hepatocellular Carcinoma And Its Implication In Hepatocarcinogenesis

OBJECTIVE:Hepatocellular carcinoma (HCC) is one of the severe lethal diseases in the world, and majority of which is HBV-related HCC. Since there are many people chronically infected with HBV in China, the HBV-related HCC takes a large part correspondingly. During the long course of being infected with HBV, the studies so far have shown that HBV DNA could integrate into human being's genome and effect the adjacent genes, which leads to the functional change and instability of human being's genome, but the precise mechanisms between the integration of HBV DNA and hepatocarcinogenesis is still unknown. The purpose of this study is to find novel integration sites of HBV DNA into human being's genome, and preliminarily explore the mechanisms of HBV DNA and hepatocarcinogenesis.METHODS:We studied HBV-related hepatocellular carcinoma tissues, matched surrounding liver tissues from 30 cases of HCC patients,and 5 cases of normal liver tissues, genomic DNA was extracted respectively, the consistency and purity of which met the requirements after analysis by UV spectrophotometer. After the primers were designed respectively, the HBV DNA integration sites were detected by Alu-PCR combined with semi-nest PCR and touchdown PCR in hepatocellular carcinoma tissues, matched surrounding liver tissues in 30 patients and 5 cases of normal liver tissues. After confirmed by separation on agarose gels and observation by ultraviolet lamp, the PCR products were sequenced. Comparing the results of sequencing with the human genome using BLAST to determine the HBV DNA integration sites and adjacent genes, we found the novel HBV DNA integration sites, and one of them is MCM3AP, which is related to the DNA replication. Total RNA were extracted from hepatocellular carcinoma tissues, matched surrounding liver tissues and normal liver tissues respectively, the consistency and purity of which met the requirements after analysis by UV spectrophotometer. After the primers were designed, we analyzed the expression levels of MCM3AP genes hepatocellular carcinoma tissues and matched surrounding liver tissues in the same sample and within the same pathological types of different hepatocellular carcinoma tissues by RT-PCR, all of which were controlled by the normal liver tissues. Confirmed by separation on agarose gels and observation by ultraviolet lamp, we preliminarily explored the effects of upgraded expression of MCM3AP on hepatocarcinogenesis.RESULTS:HBV DNA into hepatocellular carcinoma tissues were obtained by RT-PCR, sequencing. These genes encoding proteins were: LOC51030, LOC157777, minichromosome maintenance complex component 3 associated protein (MCM3AP), MCTP1, SH3 and multiple ankyrin repeat domains 2 isoform 2, CCDC40, similar to HCG2033532, mitochondrial ribosomal S5 pseudogene 4. One of them was integrated into the intron of MCM3AP. RT-PCR demonstrated that the expression levels of MCM3AP mRNA in hepatocellular carcinoma tissues, matched surrounding liver tissues and normal liver tissues are in descendent order, the ratio of MCM3AP mRNA to the GAPDH mRNA was 1.07375,0.21573,0.06747 respectively, the differences among different groups were statistically significant.(P<0.05). Meanwhile, the expression levels of MCM3AP mRNA from hepatocellular carcinoma tissues in which HBV DNA integrated into MCM3AP were still significant higher than those without HBV DNA integrated into MCM3AP.CONCLUSIONS:The HBV DNA integration sites into human genome are random, and MCM3AP is a new site. The up-regulated MCM3AP mRNA may affect flanking sequences which promote the hepatocellular carcinogenesis.