下调肌动蛋白丝相关蛋白1相似蛋白2表达对人肝癌Hep3B细胞增殖及凋亡的影响

Actin filament-associated protein 1-like 2 influence proliferation and apoptosis of human hepatocellular cancer cell Hep3B

目的观察肌动蛋白丝相关蛋白1相似蛋白2在人肝细胞癌细胞中的表达及下调后对增殖生存的影响并探讨其机制。方法以蛋白免疫印迹法(Western blot)及实时定量逆转录聚合酶链反应法(q RT-PCR)检测肝细胞癌细胞株中肌动蛋白丝相关蛋白1相似蛋白2表达,并采用小干扰RNA转染Hep3B细胞株对肌动蛋白丝相关蛋白1相似蛋白2表达进行下调,Western blot和q RT-PCR检测转染效率、增殖凋亡相关基因表达;甲基噻唑基四唑检测细胞增殖,流式细胞术检测细胞周期及凋亡。结果肌动蛋白丝相关蛋白1相似蛋白2蛋白及m RNA在肝癌细胞株Hep3B、Huh7、Hep G2、MHCC97H中均有表达,在Hep3B细胞株中表达量最高(F=5.742,P<0.01;F=3.452,P<0.05);与空白对照(si NC)组比较,si肌动蛋白丝相关蛋白1相似蛋白2组中P-P21及磷酸化的蛋白激酶B蛋白表达下调(t=8.462,P<0.01;t=9.742,P<0.01),P21、蛋白激酶B及Cleaved Capase-9蛋白表达上调(t=12.247,P<0.001;t=6.452,P<0.01;t=11.634,P<0.001;t=12.273,P<0.001);与si NC组比较,在72 h和96 h,si肌动蛋白丝相关蛋白1相似蛋白2表达下调后Hep3B细胞增殖能力下降(t=8.176,P<0.01;t=2.246,P<0.05),Hep3B停留在G1期比例增高,G_2及S期比例减少(t=4.23,P<0.05;t=2.13,P<0.05;t=5.72,P<0.05),凋亡率升高(t=8.633,P<0.01)。结论下调肌动蛋白丝相关蛋白1相似蛋白2表达通过P21磷酸化及磷脂酰肌醇3激酶/蛋白激酶B信号通路抑制肝细胞癌细胞增值及生存,可作为肝细胞癌治疗的靶向候选基因。

Objective To observe the expression of XB130 in human hepatocellular cells and the effect of XB130 down-regulating on proliferation and survival. Methods Expression of XB130 in human hepatocellular carcinoma cell-lines was detected by Western blot and （qRT）-PCR. Small interfere RNA （siRNA） was transfected into Hep3B cell to down-regulate XB130 expression and interfering effect, and proliferation and survival related gene was detected by Western blot and （qRT） -PCR. Proliferation, cell cycle and apoptosis were detected by MTF and flow cytometry. Results XB130 protein and RNA was expressed in Hep3B, Huh7, HepG2 and MHCC97H cells, highest in Hep3B cell （F = 5.742, P 〈 0.01; F = 3.452, P 〈0.05）; Compared with siNC group, p-p21 and p-Akt protein in siXB130 group was lower （t = 8.462, P 〈 0.01; t = 9.742, P 〈 0.01）, p21, Akt, Cleaved Capase-8 and Cleaved Capase-9 protein was higher （t = 12.247, P 〈 0.01; t = 6.452, P 〈 0.01; t = 11.634, P 〈 0.01; t = 12.273, P 〈 0.01）; Compared with siNC group, at 72 h and 96 h, proliferation of Hep3B cells wad decreased （t = 8.176, P 〈 0.01; t = 2.246, P〈 0.05）, more Hep3B cells stayed at GI phase, less stayed at G2 and S phase （t = 4.23, P 〈0.05; t = 2.13, P〈 0.05; t = 5.72, P 〈 0.05）, apoptosis rate was increased （t = 8.633, P 〈 0.01）. Conclusions siXB 130 down-regulating inhibits apoptosis and survival via p-21 phosphorylation and PI3K/Akt signal pathway in hepatocellular carcinoma cells and may be a target candidate for hepatocellular carcinoma therapy.