营养缺乏状况下干扰ASPP2通过调节自噬促进肝癌细胞增殖

ASPP2 gene silence promotes hepatocellular carcinoma cell proliferation via regulating autophagy under starvation

目的观察营养缺乏状况下干扰p53凋亡刺激蛋白2(ASPP2)对肝癌细胞增殖和凋亡的影响及机制。方法构建RNA干扰抑制ASPP2的慢病毒载体表达体系并感染肝癌细胞HepG2,通过电镜观察、MTS法检测、流式细胞术检测以及形态学观察,探讨在缺乏氨基酸和血清的培养条件下干扰ASPP2表达对肝癌细胞HepG2增殖和凋亡的影响,并以自噬抑制剂3-甲基腺嘌呤(3-MA)进行干预,探讨ASPP2下调后对细胞的作用是否与自噬有关。结果在缺乏氨基酸和血清的培养系统里,电镜观察显示干扰ASPP2后自噬泡形成明显增加(P<0.05),荧光显微镜下可见GFP-LC3荧光自噬小体的表达提高(P<0.05)。MTS检测显示干扰ASPP2表达后HepG2细胞的增殖能力明显增强(P<0.05),而自噬抑制剂3-MA能够抑制其作用(P<0.05)。形态学观察发现干扰ASPP2能够提高HepG2细胞的抗凋亡能力,应用自噬抑制剂3-MA后细胞活性明显降低,出现了细胞内颗粒物增加等一系列早期死亡的症状。AnnexinⅤ-PI双染显示干扰ASPP2使细胞凋亡率由(38±5)%下降为(15±4)%(P<0.05),加入自噬抑制剂3-MA后细胞凋亡率上升至(36±3)%(P<0.05)。结论在缺乏营养的状态下,下调ASPP2可能通过调节自噬促进肝癌细胞的增殖和存活。

Objective To study the effects of apoptosis-stimulating protein of p53-2 （ASPP2） gene on hepatocellular carcinoma （HCC） cell proliferation and apoptosis under starvation and the related mechanism. Methods Lentivirus encoding shRNA against ASPP2 was constructed to 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 of HepG2 cells from （384-5）%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.