沉默热休克蛋白70-2基因经线粒体依赖通路介导肝癌细胞凋亡

Inhibition of HSP70-2 expression by RNA interference induces apoptosis of human hepatocellular carcinoma cells

目的探讨沉默热休克蛋白（HSP）70—2对肝癌细胞生长、凋亡的影响，以及诱导肝癌细胞凋亡的详细作用机制。方法Western blot、免疫细胞化学法检测HSP70—2在4种肝癌细胞和正常肝细胞中的表达。设计合成针对HSP702基因的特异性短发夹状RNA（shRNA），构建真核表达载体HSP70-2 shRNA1和HSP70—2 shRNA2。转染肝癌细胞后，四甲基偶氮唑盐法检测细胞增殖，膜联蛋白／碘化丙啶双染法检测细胞凋亡情况，罗丹明染色检测细胞线粒体跨膜电位变化，Western blot检测多聚ADP-核糖聚合酶、caspase3、caspase-9蛋白切割情况，以及细胞色素C、Bax、Bcl-2蛋白的表达变化。结果HSP702在4种肝癌细胞中均高表达，在L02细胞中仅有微量表达。HSP70-2 shRNA1、shRNA2均能有效抑制肝癌细胞中HSP70—2的表达。沉默HSP70-2基因显著抑制肝癌细胞生长，诱导肝癌细胞凋亡；转染48h后，shRNAl和shRNA2诱导HepG2细胞的凋亡指数分别为55．8％±1．8％和57．1％±1．6%；诱导Huh细胞凋亡指数分别为54．9%±1．1％和60．2％±2．6%，与对照组相比，差异均有统计学意义，P值均〈0．01。转染HSP70—2 shRNA48h后，肝癌细胞线粒体跨膜电位显著下降，细胞色素C从线粒体释放到胞质中，caspase3、caspase-9激活，多聚ADP核糖聚合酶被剪切降解，抗凋亡蛋白Bcl-2表达减少，促凋亡蛋白Bax表达明显增加。结论沉默HSP70-2能通过激活线粒体凋亡通路导致肝癌细胞凋亡。

Objectives To determine the effect of short hairpin RNA targeting HSP70-2 on the growth and apoptosis of hepatocellular carcinoma （HCC） cells, and to elucidate its possible mechanisms in mitochondria apoptotie pathway. Methods Western blot and immunocytochemistry were used to determine the expression of HSP70-2 in HCC cells and normal hepatocytes. HepG2 and Huh-7 cells were cultured and transfected with HSP70-2 shRNA1 and shRNA2. Cell proliferation was examined by MTT. Cell apoptosis and mitochondria membrane potential were determined by flow cytometery. Western blot was used to analyze the expressions of apoptosis related proteins including Bax, Bcl-2, PARP, caspase-9 and caspase-3. Results HSP70-2 was expressed at high levels in hepatocellular carcinoma （HCC） cells （SNU-449, HepG2, Huh-7, Hep3B） whereas there were very low levels in normal hepatocytes （L02）. Using DNA vector-based RNA interference, we found that knockdown of HSP70-2 inhibited the growth of HCC cells through induction of mitochondria-dependent apoptosis. The mitochondria-dependent apoptosis induced by HSP70-2 knockdown was indicated by cytochrome c release from mitochondria, activation of caspase-9 and caspase-3, and loss of mitochondrial potential. Furthermore, knockdown of HSP70-2 resulted in up-regulation of the pro-apoptotic factor Bax and down-regulation of the pro-survival factor Bcl-2. Conclusions Our results indicated that HSP70-2 down-regulation induces apoptosis of HCC cells through the mitochondrial apoptotic pathway, highlighting the importance of HSP70-2 in survival of HCC cells and maintenance of liver function.