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

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 apoptotic 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 cytometry. 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.