自噬基因Beclin 1 shRNA表达质粒的构建及其下调凋亡因子caspase-9效应的研究

Construction of shRNA Expression Vectors for Autophagy Gene Beclin 1 and Their Downregulation Effect on Caspase-9

构建自噬基因Beclin 1的小发夹RNA(shRNA)真核表达质粒,脂质体包裹后体外转染人宫颈癌HeLa细胞株,通过荧光定量PCR(RT-PCR)和Western blot检测其对HeLa细胞自噬基因Beclin 1 mRNA及蛋白表达的影响,并检测细胞增殖、细胞周期、细胞凋亡情况以及凋亡因子caspase-9 mRNA及蛋白表达的变化。结果表明shRNA真核表达载体可以使HeLa细胞中自噬基因Beclin 1的mRNA及其蛋白含量降低,转染后细胞生长增殖速度加快,凋亡率降低,并伴有caspase-9 mRNA及蛋白量的显著下调。因此,Beclin 1不仅与自噬调控通路有关,而且可以调控凋亡的发生,同时参与两种程序性细胞死亡的过程。

The shRNA expression vectors were constructed and transfected via lipofectamine into HeLa cells. Real time-ploymerase chain reaction （RT-PCR） and Western blot were used for detecting the expression of mRNA and protein of Beclinl in transfected cells. Flow cytometry was employed to observe the effect of transfection on the apoptosis and cell cycle of HeLa, and proliferation was analyzed by MTT assay. The expression of caspase-9 in transfection cells was also detected by RT-PCR and Western blot. The constructed vectors significantly inhibited the expressin of mRNA and the protein of Beclinl in HeLa cells. The growth of transfected cells was promoted, and less apoptosis cells were identified in these cells. After transfection of the constructed vectors into HeLa cells, the expression of caspase-9 was effectively inhibited. All of these indicate that autophagy and apoptosis are two types of programmed cell death, that autophagy gene Beclin 1 plays an important role in these two types, and that defect of autophagy and apoptosis may be important in tumor genesis.