ASPP2 gene silence promotes hepatocellular carcinoma cell proliferation via regulating autophagy under starvation / 第二军医大学学报

ABSTRACT

Objective To study the effects of apoptosis-stimulating protein of ρ53-2 (ASPP2) gene on hepatocellular carcinoma (HCC) cell proliferation and apoptosis under starvation and the related mechanism. Methods Lentivirus encoding shRNA against ASPP2 was constructedto knockdown ASPP2 expression in hepatoma cell HepG2. Cell proliferation and apoptosis were observed by transmission electron microscopy, MTS analysis, flow cytometry analysis and morphologic changes. The influence of silencing ASPP2 gene on the proliferation and apoptosis under amino acid-starvation and serum-deprivation culture was observed; autophagy inhibitor 3-methyladenine (3-MA) was added in the experiment so as to detect the involvement of autophagy in the changes induced by ASPP2 down-regulation. Results Transmission electron microscopy showed cytoplasmic accumulation of autophagosomes when ASPP2 was knocked down under amino acid-starvation and serum- deprivation (P<0. 05), with increased GFP-LC3 dots (P<0. 05). MTS analysis showed that silence of ASPP2 gene greatly enhanced the proliferation of HepG2 cells (P<0. 05), which could be inhibited by addition of 3-MA (P<0. 05). Microscope observation showed that silence of ASPP2 gene promoted the anti-apoptotic ability of HepG2 cells, which was reversed by treatment with 3-MA. Early sign of apoptosis was observed in shASPP2 + 3-MA group. Annexin V_PI double staining showed that ASPP2 silence decreased the apoptotic rate ofHepG2 cells from (38±5)% to(15±4)% (P<0. 05), and 3-MA treatment increased it to (36 ±3)% (P<0. 05). Conclusion Down-regulation of ASPP2 expression may facilitate the survival and proliferation of HCC cells through activating autophagy under starvation.