RNA干扰Ref-1表达对肝癌顺铂敏感性的影响

Effect of expression of redox factor-1 mediated by RNA interference on sensitivity to cisplatin in human hepatoma

目的建立表达氧化还原因子-1(Ref-1)小分子干扰RNA的重组逆转录病毒载体pmscv/siRef-1,观察人肝癌细胞株HepG2下调内源性Ref-1后对顺铂的敏感性变化。方法采用基因重组技术构建逆转录病毒载体pmscv/siRef-1。包装逆转录病毒,感染HepG2。RT-PCR及Western blot法检测感染后细胞内Ref-1 mRNA和蛋白表达,MTT法检测HepG2细胞下调Ref-1表达后对顺铂的敏感性变化。结果pmscv/siRef-1能有效被包装并感染靶细胞,感染pmscv/siRef-1病毒的HepG2细胞Ref-1蛋白与mRNA表达量明显降低,细胞对顺铂敏感性显著增加(P<0.05)。结论成功构建以逆转录病毒为基础的抑制内源性Ref-1表达的RNA干扰载体;RNA干扰Ref-1基因可显著提高HepG2细胞对顺铂的敏感性。

Objective To construct and identify the recombinant retreviral vectors expressing siRNA for Ref-1 gene kneck-down, and sequentially to detect the sensitivity of HepG2/siRef-1 cells to cisplatin. Methods Recombinant retrovirus plasmid pmscv/siRef-1 was constructed, and transfected into HepG2 cells. RT-PCR and Western blot were performed to measure the expression of Ref-1 mRNA and protein, respectively. The proliferations of HcpG2/siRef-1 cells in response to cisplatin were detected by MTT assay. Results The pmscv/siRef-1 were successfully packaged and transfected to the HepG2 cells. The level of Ref-1 mRNA and protein were significantly reduced in HepG2 cells infected with Ref-1 siRNA（P 〈0.05 ）. Rcf-1 down-regulation suppressed the proliferation of HepG2 cells under cisplatin treatment. Conclusion The recombinant vector pmscv/siRef-1 can be successfully constructed. The silencing of Ref-1 mediated by RNA interference can increase the sensitivity of HepG2 cells to cisplatin.