沉默FABP-5基因对人肝癌HepG2细胞的影响

Effect of RNA interference targeting FABP-5 on cell proliferation, apoptosis and invasion in human hepatocellular carcinoma cell line HepG2

目的:研究RNA干扰技术的重组慢病毒载体沉默人肝癌Hep G2细胞中表皮脂肪酸结合蛋白-5(fatty acid binding protein-5,FABP-5)基因对其增殖、凋亡和侵袭力的影响及作用机制.方法:构建3个靶向FABP-5基因sh RNA载体,并筛选出最有效的靶点.将Hep G2细胞分为3组:实验组用FABP-5基因沉默重组慢病毒颗粒(LV-sh RNA-FABP-5)感染HepG2细胞;阴性对照组用空载慢病毒颗粒(LV-sh RNANC组)感染Hep G2;空白对照组正常培养.应用RT-PCR、实时荧光定量PCR检测FABP-5基因m RNA的表达;Western blot技术检测各组细胞FABP-5蛋白的相对表达;MTT法测定细胞体外增殖能力;Giemsa染色法检测细胞的克隆形成;细胞侵袭小室法检测各组细胞的体外侵袭力;流式细胞技术(flow cytometry,FCM)检测各组细胞增殖和凋亡的变化情况.结果:通过测序验证构建的FABP-5-shRNA表达载体,感染人肝癌HepG2细胞,荧光显微镜观察到细胞感染率>90%.Real-time PCR和Western blot结果得出:相比空白对照组和阴性对照组,实验组的3种靶点FABP-5-sh RNA干扰序列中,LV-sh RNA-FABP-5(1)靶点中FABP-5表达的敲减率最高(P<0.05),筛选出此组为最佳靶点;MTT法结果提示:实验组细胞490 n m处的吸光度(A)值在转染后第1-5天时均低于阴性对照组、空白对照组,差别有统计学意义(P<0.05).Giemsa染色法结果显示:稳定转染Hep G2细胞后细胞的增殖能力明显下降(P<0.05);细胞侵袭小室实验显示:与空白对照和阴性对照组相比,实验组细胞的侵袭力明显受到抑制(P<0.05);流式细胞技术检测发现实验组的细胞相对于阴性对照组出现了明显的凋亡(P<0.05).实验组较阴性对照组、空白对照组G2/M期延长,G1期缩短,差别具有统计学意义(P<0.05).结论:人肝癌Hep G2细胞中沉默FABP-5基因可以有效抑制其表达,能够降低肝癌细胞的侵袭力,抑制肝癌细胞的增殖,将细胞周期阻滞于G2/M期,使其凋亡显著增加.

AIM： To investigate the effect of recombinant lentiviral mediated RNA interference（RNAi） targeting the fatty acid binding prote in- 5（FABP-5） gene on cell proliferation, apoptosis and invasiveness in human hepatocellular carcinoma cell line HepG2, and to explore the possible underlying mechanisms. METHODS ： Three vectors carrying shorthairpin RNAs（sh RNAs） targeting the FABP-5 gene（ FABP- 5- shRNA expression vectors） were constructed and selected for the most effective one. HepG2 cells were divided into three groups： an experimental group, a normal control group and a negative control group. For the experimental group, HepG2 cells were transfected with the recombinant lentiviral vector（LV-shR NA-FABP-5）, the negative control group was transfected with a control lentiviral vector（LV-shR NA-NC）, and the normal control group did not undergo any treatment. The mR NA level of FABP-5 was analyzed by reverse transcriptionp olymeras chain reaction（ R T- P C R） a n d quantitative real-time polymerase chain reaction（qP CR）. The relative expression of FABP-5 protein was analyzed by Western blot. Cell proliferation was detected by MTT assay, cell colony formation was detected by Giemsa staining, cell invasion ability was assessed using the cell invasion chamber method, and cell cycle and apoptosis were observed by flow cytometry（FCM）. RESULTS： FABP-5-sh RNA expression vectors w e retrans fected into HepG2cells, and fluorescence analysis indicated that 〉90% of cells showed fluorescence signal. Compared with the normal control group and negative control group, FABP-5 m RNA and protein expression was significantly down-regulated in cells transfected with the three shR NA carrying vectors, with the LV-shR NA-FABP-5 having the highest efficiency（P 〈0.05）. HepG2 cell transfected with the FABP-5-shR NA had significantly reduced proliferation and invasion compared with the other two groups（P〈 0.05）. Flow cytometry analysis showed that the experimental group showed obvious apoptosis（P 〈0.05）. The percentage of cells in G2/M phase significantly increased in cells transfected with the FABP-5-sh RNA（P 〈0.05）. CONCLUSION： The high expression of the FABP-5 gene could be silenced by RNAi, and RNA i- induced FABP- 5 knock down could effectively inhibit the proliferation and invasion of Hep G2 cells, block the cell cycle in G2/M phase, and significantly increase cell apoptosis.