人BNIP3真核表达载体和shRNA表达载体的构建及其对肝癌细胞自噬的影响

Effects of BNIP3 on autophagy of hepatoma cells:a study using BNIP3 over-expression vectors and BNIP3-shRNA vectors

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 explore the effects of BNIP3 on autophagy of hepatoma cells by using over-expression vectors and shRNA vectors. Methods BNIP3 cDNA was extracted from human hepatoma cell line BEL-7402 and was cloned into the pcDNA3.1-His-C vector to construct an over-expression vector（pcDNA3.1-BNIP3 ）. The recombinant shRNA expression vector targeting the BNIP3 gene was also constructed （pSliencer-BNIP3）. Both recombinant plasmids were verified by sequencing, enzymatic digestion, RT-PCR and Western blot. The effects of BNIP3 on autophagy of hepato- ma cells were investigated by transfecting BEL-7402 hepatoma cells with the recombinant plasimids. Results Autoph- agy of hepatoma cells was significantly enhanced with pcDNA3. 1-BNIP3 and compromised with pSilencer-BNIP3. Conclusion BNIP3 was involved in promoting autophagy of hepatoma cells.