丙型肝炎病毒非结构蛋白5A下调抑癌基因PTEN的表达

Nonstructural protein 5A of hepatitis C virus down-regulates the expression of PTEN

目的萤虫素酶探讨丙型肝炎病毒非结构蛋白5A(HCVNS5A)对抑癌基因PTEN表达的影响。方法表达HCVNS5A的质粒(pCNS5A)转染QSG-7701细胞,采用间接免疫荧光法观察HCVNS5A蛋白的表达。构建PTEN启动子驱动的萤虫素酶报告基因表达质粒PTEN-Luc,并与pCNS5A共转染QSG-7701细胞,萤虫素酶实验检测HCVNS5A对PTEN启动子活性的影响。采用RT-PCR和Westernblot分析分别观察HCVNS5A对PTENmRNA和PTEN蛋白表达水平的影响。结果转染pCNS5A的QSG-7701细胞胞浆中可见HCVNS5A蛋白表达。PTEN-Luc与不同剂量的pCNS5A质粒共转染QSG-7701细胞,HCVNS5A表达组的PTEN-Luc相对活性下降,且转染HCVNS5A质粒的剂量越大,PTEN-Luc的相对活性越低。转染pCNS5A的QSG-7701细胞内PTENmRNA和蛋白质表达均较对照组下降,且随着HCVNS5A质粒剂量增大,PTENmRNA和PTEN蛋白表达下降更明显。结论 HCVNS5A通过抑制PTEN启动子的活性而抑制PTEN基因的转录和PTEN蛋白的表达,这可能是HCV所致原发性肝细胞癌的发病机制之一。

Objective To explore whether the non-structural protein 5A （NSSA） encoded by human hepatitia C virus （HCV） RNA genome could affect PTEN expression. Methods HCV NSSA expressing plasmid was transfected into QSG-7701 cells. Indirect immunofluorescence was applied to detect the expression of HCV NSSA in theses cells. The PTEN promoter reporter plasmid PTEN-Luc was cloned by PCR from human genomic DNA. The PTEN-Luc plasmid was transfected alone or co-transfected with HCV NSSA expressing plasmid pCNSSA into QSG-7701 cells. Luciferase assay were carried out. PTEN mRNA transcript and protein levels were assessed by RT-PCR and Western blot, respectively. Results HCV NSSA protein was detected in the cytoplasm of QSG-770! cells transfected with pCNSSA. The relative luciferase activity of PTEN-Luc was down-regulated in the presence of HCV NSSA protein, with dose-dependent manner. The PTEN mRNA level was also down-regulated in cells transfected with pCNSSA plasmid, and PTEN protein was significantly inbibitted by HCV NSSA protein. The repression of PTEN mRNA and protein by HCV NSSA was also dose-dependent. Conclusions HCV NSSA down-regulates PTEN expression at transcriptional level by inhibitting the promoter activity, mRNA transcription and protein levels. The data shine a new light on the contributory role of NSSA in HCV-related hepatocellular carcinoma.