Kordestani Roghyeh, Mirshafiee Hamideh, Hosseini Seyed Masoud, Sharifi Zohreh
Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran ; Department of Microbiology, School of Biological Science, Shahid Beheshti University, Tehran, Iran.
Department of Microbiology, School of Biological Science, Shahid Beheshti University, Tehran, Iran.
Avicenna J Med Biotechnol. 2014 Jan;6(1):3-9.
The HBV-X (HBX) protein is believed to contribute to the development of HCC. However, the molecular mechanisms involved in HBX- mediated hepatocarcinogenesis remain obscure. In this study, the effect of hepatitis B virus X gene and its protein product HBxAg on expression of p53 gene in Hep G2 cell line was investigated.
Viral DNA extracted from HBV-positive serum and HBX gene region was amplified using polymerase chain reaction (PCR). Then, PCR product was cloned into the pcDNA3 vector. After confirmation of cloning, the recombinant plasmid pcDNA3-X was transfected into HepG2 cell line using lipid-mediated DNA-transfection procedure. SDS-PAGE and western blotting methods were used to identify expression of HBX protein. Relative quantification was used to analyze the p53gene expression using the 2-(ΔΔ Ct) method.
Recombinant plasmid pcDNA3-HBX was confirmed by restriction endonucleases digestion and colony-PCR. The results of SDS-PAGE and western blot assays showed that HBX gene could be expressed in Hep G2 cell line. There was no significant difference between the expression levels of p53 compared with GAPDH gene as housekeeping gene (p < 0.05).
There was no significant difference in the protein levels between the transfected cells with X gene containing HBX130 and HBX131 double mu-tations and p53 gene. It is necessary to do more studies on Hepatitis B virus to understand the role of HBX on the development of liver cancer and its function on p53 tumor suppressor protein.
乙型肝炎病毒X（HBX）蛋白被认为与肝癌的发生发展有关。然而，HBX介导的肝癌发生的分子机制仍不清楚。本研究探讨了乙型肝炎病毒X基因及其蛋白产物HBxAg对Hep G2细胞系中p53基因表达的影响。
从HBV阳性血清中提取病毒DNA，采用聚合酶链反应（PCR）扩增HBX基因区域。然后，将PCR产物克隆到pcDNA3载体中。克隆确认后，采用脂质介导的DNA转染方法将重组质粒pcDNA3-X转染到HepG2细胞系中。采用SDS-PAGE和蛋白质印迹法鉴定HBX蛋白的表达。采用2-(ΔΔCt)法进行相对定量分析p53基因表达。
通过限制性内切酶消化和菌落PCR对重组质粒pcDNA3-HBX进行了确认。SDS-PAGE和蛋白质印迹分析结果显示，HBX基因可在Hep G2细胞系中表达。与看家基因GAPDH相比，p53的表达水平无显著差异（p<0.05）。
含有HBX130和HBX131双突变的X基因转染细胞与p53基因在蛋白水平上无显著差异。有必要对乙型肝炎病毒进行更多研究，以了解HBX在肝癌发生中的作用及其对p53肿瘤抑制蛋白的功能。
