DIS3通过调控PI3K/AKT/mTOR信号通路促进人骨髓瘤细胞凋亡

DIS3 promotes apoptosis of human myeloma cells by regulating PI3K/AKT/mTOR signaling pathway

目的探究DIS3对人骨髓瘤细胞凋亡的影响及其作用机制。方法选取人骨髓瘤细胞株NCI-H929、RPMI 8226和U266为研究对象,分别设计并构建DIS3基因过表达载体和DIS3-siRNA,3种细胞均随机分成5组:对照组、DIS3-siRNA阴性对照(negative control,NC)组、DIS3空载组、DIS3过表达组以及DIS3-siRNA组。细胞培养48 h后,流式细胞术检测细胞凋亡水平。qRT-PCR及Western blot检测凋亡相关基因Bax、Bcl-2及Caspase-3在mRNA和蛋白水平上的表达,同时检测PI3K、AKT及mTOR的表达情况。结果NCI-H929、RPMI 8226和U266细胞转染后,与DIS3空载组比较,DIS3过表达组细胞凋亡率均显著上升(P<0.01),Bax、Caspase-3、PI3K、AKT和mTOR的表达水平显著升高(P<0.05或P<0.01),Bcl-2的表达水平显著降低(P<0.05或P<0.01)。与DIS3-siRNA NC组比较,DIS3-siRNA组NCI-H929、RPMI 8226和U266细胞凋亡率均显著下降(P<0.05或P<0.01),Bax、Caspase-3、PI3K、AKT和mTOR的表达水平显著降低(P<0.05或P<0.01),Bcl-2的表达水平显著升高(P<0.05或P<0.01)。结论DIS3过表达能显著促进人骨髓瘤细胞凋亡的发生,促进Bax和Caspase-3表达而抑制Bcl-2的表达,这可能与对PI3K/AKT/mTOR信号通路的调控密切相关。

Objective To investigate the effects of DIS3 on apoptosis of human myeloma cells and its mechanism. Methods Human myeloma cell lines NCI-H929, RPMI 8226 and U266 were selected for the study. DIS3 gene overexpression vectors and DIS3-siRNA were designed and constructed, respectively. All three kinds of cells were randomly divided into five groups: control group, DIS3-siRNA negative control(NC) group, DIS3 empty vector group, DIS3-overexpression group and DIS3-siRNA group, respectively. After 48 h cell culture, the apoptosis rate was detected by flow cytometry. RT-qPCR and Western blot were performed to detect the expression of apoptosis-related genes Bax, Bcl-2 and Caspase-3 and the expression of PI3 K, AKT and mTOR at the mRNA and protein levels. Results Compared with DIS3 empty vector group, the apoptosis rates of NCI-H929, RPMI 8226 and U266 cells were significantly increased in DIS3 overexpression group(P<0.01), the expression levels of Bax, Caspase-3, PI3 K, AKT and mTOR were significantly also increased(P<0.05 or P<0.01), while the expression level of Bcl-2 was significantly decreased(P<0.05 or P<0.01). Compared with DIS3-siRNA NC group, the apoptosis rates of NCI-H929, RPMI 8226 and U266 cells were significantly decreased in DIS3-siRNA group(P<0.05 or P<0.01), the expression levels of Bax, Caspase-3, PI3 K, AKT and mTOR were significantly decreased(P<0.05 or P<0.01), while the expression level of Bcl-2 was significantly increased(P<0.05 or P<0.01). Conclusion DIS3 overexpression significantly promotes the apoptosis of human myeloma cells, the expression of Bax and Caspase-3, and suppresses the expression level of Bcl-2, which may be closely related to the regulation of PI3 K/AKT/mTOR signaling pathway.