RNAi介导BMPR-Ⅱ基因沉默对人肝癌HepG2细胞增殖和侵袭的影响

Effect of siRNA interference targeting BMPR-Ⅱ gene on cell proliferation and invasion ability of human hepatoma HepG2 cells

背景与目的:原发性肝细胞癌是我国常见的恶性肿瘤之一,近期研究发现骨形态发生蛋白(bone morphogenetic proteins,BMPs)信号通路在肿瘤的发生发展中发挥重要作用。本研究采用RNA干扰技术(RNA interference,RNAi)抑制骨形态发生蛋白受体Ⅱ(bone morphogenetic protein receptor,BMPR-Ⅱ)表达,观察抑制效果及抑制BMPR-Ⅱ表达后对人肝癌HepG2细胞增殖和侵袭的影响。方法:设计并化学合成针对BMPR-Ⅱ的3对小干扰RNA(small interfering RNA,siRNA),阳离子脂质体法瞬间转染HepG2细胞。采用离体培养肝癌HepG2细胞,并设正常对照组、空白对照组、阴性siRNA组及siRNA-BMPR-Ⅱ-a、siRNA-BMPR-Ⅱ-b、siRNA-BMPR-Ⅱ-c转染组。各组培养48 h后,运用半定量逆转录聚合酶链反应(RT-PCR)和Western blot法检测HepG2细胞中BMPR-Ⅱ在mRNA和蛋白水平表达的变化,MTT比色法及Transwell侵袭实验检测转染后细胞增殖及侵袭的变化。结果:RT-PCR、Western blot显示,3个特异性siRNA-BMPR-Ⅱ转染组转染48 h后,其BMPR-Ⅱ的mRNA和蛋白表达与另外3组比较受到明显抑制,以siRNA-BMPR-Ⅱ-a转染组抑制效果最明显(0.70±0.06、0.45±0.10,P<0.05)。MTT比色法及Transwell侵袭实验显示,转染48 h后,siRNA-BMPR-Ⅱ-a转染组HepG2细胞的生长及穿膜数(48.27%±0.76%、25.20±1.60,P<0.05)明显低于正常对照组(82.64%±0.67%、60.40±1.39)及阴性对照组(81.21%±0.80%、59.50±1.85)。结论:siRNA干扰介导BMPR-Ⅱ基因沉默可抑制人肝癌HepG2细胞的增殖和侵袭,BMPR-Ⅱ可作为肝癌抗肿瘤治疗的新靶点。

Background and purpose：Primary hepatocellular carcinoma is one of the most common malignant tumors in our country.Recent studies showed that bone morphogenetic proteins（BMPs） signaling pathways play a key role in the development of tumors.This study aimed to investigate the effect of small interfering RNA（siRNA） on the inhibiting of bone morphogenetic protein receptor（BMPR-Ⅱ）,and to observe its effect on the proliferation and invasion of human hepatoma HepG2 cells after BMPR-Ⅱ silencing.Methods：Three pairs of specific siRNA targeting BMPR-Ⅱ were designed and synthesized,and then transiently transfected into HepG2 cells via cathodolyte liposome transfection method.In vitro cultured HepG2 cells were divided into a normal control group,a blank control group,a negative control group and 3 transfected with siRNA-BMPR-Ⅱ-a,siRNA-BMPR-Ⅱ-b,siRNA-BMPR-Ⅱ-c groups.RT-PCR and Western blot were used to detect BMPR-Ⅱ expression at mRNA and protein levels,and the proliferation and invasion ability of HepG2 cells after transfection were assessed by MTT assay and Transwell invasion assay respectively.Results：Forty-eight hours after the 3 pairs of specific siRNA-BMPR-Ⅱ were transfected into HepG2 cells,RT-PCR and Western blot results showed that the expression of BMPR-Ⅱ mRNA and protein were significantly inhibited compared with those in the other 3 groups,with siRNA-BMPR-Ⅱ-a having the highest inhibitory rate（0.70±0.06,0.45±0.10,P0.05）.MTT assay and Transwell invasion assay demonstrated the growth and the number of HepG2 cells in siRNA-BMPR-Ⅱ-a group was significantly lower（48.27%±0.76%,25.20±1.60,P0.05） than in normal control group（82.64%±0.67%,60.40±1.39） and negative control group（81.21%±0.80%,59.50±1.85） at 48 h after transfection.Conclusion：siRNA interference-mediated BMPR-Ⅱ gene silencing can inhibit the proliferation and invasion ability of human HepG2 cells,and BMPR-Ⅱ gene may be used as a new promising target of antineoplastic therapy in hepatocellular carcinoma.