微小RNA-132转染对体内外肝癌生长和凋亡的作用

Effects of MicroRNA-132 Transfection on the Proliferation and Apoptosis of Human Liver Cancer Cells in vitro and in vivo

目的观察转染微小RNA-132(miR-132)对体内外人肝癌细胞株MHCC97H生长和凋亡的影响,初步探讨其作用机制。方法采用实时荧光定量逆转录-聚合酶链反应(RT-q PCR)检测45例肝癌组织及癌旁正常组织中miR-132的表达,CCK-8法、流式细胞术、裸鼠体内成瘤实验和TUNEL实验检验转染miR-132后对体内外MHCC97H细胞生长和凋亡的作用,Western blot法检测体外MHCC97H细胞中p-AKT、Survivin和Caspase 3蛋白的表达,免疫组织化学法检测体内肿瘤组织中Ki-67、Survivin和Caspase 3的表达。结果 miR-132在肝癌组织中的表达明显低于癌旁正常组织(P<0.05)。在体外实验中,与空白对照组和阴性对照组相比,转染组细胞中miR-132的表达明显升高,细胞増殖明显被抑制,G0/G1期细胞比例明显上升,S期细胞比例明显下降,凋亡细胞显著增加(P均<0.05)。转染组细胞中Survivin和p-AKT的表达下调,Caspase 3的表达上调,与空白对照组和阴性对照组相比,差异均有统计学意义(P均<0.05)。在体内实验中,转染组裸鼠皮下移植瘤生长明显被抑制,凋亡肿瘤细胞明显增加,Survivin和Ki-67蛋白表达下调,而Caspase 3表达增加(P均<0.05)。结论转染miR-132对体内外肝癌细胞有增殖抑制和凋亡诱导作用,miR-132有望成为肝癌治疗的新靶点。

Objective To observe the biological role and underlying mechanism of microRNA-132（ miR-132） in liver cancer cell proliferation and apoptosis. Methods The expressions of miR-132 in the cancer tissue and their adjacent tissues from 45 liver cancer patients were detected by real-time quantitative polymerase chain reaction（ RT-q PCR）. The biological effects of miR-132 transfection on human liver cancer MHCC97 H cells were assessed by CCK-8 assay,flow cytometry,in vivo experiment in nude mice,and TUNEL test. Western blotting was used to detect the expressions of p-AKT,Survivin,and Caspase 3 in liver cancer cells. Immunohistochemistry was used to detect the positive expressions of Ki-67,Survivin,and Caspase 3 in the xenograft tumors. Results The expression level of miR-132 was found to be down-regulated in liver cancer tissues compared with the matched adjacent tissues（ P 0. 05）. After transfection,the expression of miR-132 was significantly higher than blank control group and negative control group（ P 0. 05）. The proliferation of liver cancercells was inhibited significantly by miR-132 transfection（ P 0. 05）. Transfection of miR-132 arrested cells in the G0/ G1 phase and triggered apoptosis of MHCC97 H cells（ P 0. 05）. After miR-132 transfection,the expression of Caspase 3 was up-regulated, whereas the expressions of p-AKT and Survivin were down-regulated（ P 0. 05）. In addition,the tumor weight in miR-132 transfection group was significantly decreased in comparison with blank control group and negative control group（ P 0. 05）. Apoptosis occurred more frequently in the miR-132 transfection group than in control groups（ P 0. 05）. Compared with the blank control group and negative control group,the miR-132 transfection group had significantly decreased expression of Survivin but increased positive expression of Ki-67 and Caspase 3（ P 0. 05）. Conclusions miR-132 is down-regulated in human liver cancer tissues. miR-132 transfection can effectively inhibit proliferation and promote apoptosis of MHCC97 H cells in vitro and in vivo. Therefore,miR-132 may become a new target in liver cancer treatment.