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目的利用人BNIP3的真核表达载体和靶向BNIP3的shRNA表达载体,研究BNIP3过表达和封闭时对肝癌细胞自噬的影响。方法从人肝癌细胞系中,通过RT-PCR扩增人BNIP3基因编码区序列,酶切后插入pcDNA3.1-His-C载体,以此构建重组pcDNA3.1-BNIP3真核表达载体。同时构建靶向人BNIP3基因的shRNA表达载体pSilencer-BNIP3。分别经测序、酶切、RT-PCR和Western blot等方法对重组载体是否构建成功进行验证。并通过采用Western blot检测自噬标志物LC3蛋白的剪切,研究BNIP3过表达和抑制时对肝癌细胞自噬的影响。结果pcDNA3.1-BNIP3和pSilencer-BNIP3分别过表达BNIP3和抑制BNIP3的表达。BNIP3过表达能够显著增加肝癌细胞的自噬;BNIP3表达受到抑制时,肝癌细胞的自噬明显减少。结论成功构建了人BNIP3的真核表达载体pcDNA3.1-BNIP3和shRNA表达载体pSilencer-BNIP3,并在人肝癌细胞系中证实BNIP3对肝癌细胞自噬的促进作用。
Objective To study the effects of BNIP3 overexpression and blocking on the autophagy of hepatocellular carcinoma cells by using the eukaryotic expression vector of human BNIP3 and the shRNA expression vector targeting BNIP3. Methods The coding region of human BNIP3 gene was amplified from human hepatocellular carcinoma cell line by RT-PCR and inserted into pcDNA3.1-His-C vector after digestion. The eukaryotic expression vector pcDNA3.1-BNIP3 was constructed. At the same time, a shRNA expression vector pSilencer-BNIP3 targeting human BNIP3 gene was constructed. The successful construction of the recombinant vector was verified by sequencing, digestion, RT-PCR and Western blot respectively. The effect of BNIP3 overexpression and inhibition on the autophagy of hepatocarcinoma cells was studied by Western blot analysis of the cleaved LC3 protein. Results pcDNA3.1-BNIP3 and pSilencer-BNIP3 overexpression BNIP3 and BNIP3 expression. BNIP3 overexpression can significantly increase the autophagy in liver cancer cells; when BNIP3 expression is inhibited, the autophagy of liver cancer cells is significantly reduced. Conclusion The eukaryotic expression vector pcDNA3.1-BNIP3 of human BNIP3 and the shRNA expression vector pSilencer-BNIP3 were successfully constructed. The pro-apoptotic effect of BNIP3 on hepatocarcinoma cell autophagy was confirmed in human hepatoma cell line.