慢病毒介导的mTOR敲低肝癌细胞株HepG2的构建及初步功能检测

目的:建立高效稳定的哺乳动物雷帕霉素靶蛋白(mTOR)小干扰RNA(siRNA)细胞导入方法,并对mTOR敲低的HepG2肝癌细胞株的功能进行初步检测。方法:构建了2条不同的人mTOR慢病毒siRNA载体pLenti-H1/mTOR siRNA,与3个包装质粒共转染293T细胞,包装成慢病毒后感染HepG2细胞;经嘌呤霉素筛选2周后,收集细胞进行Western印迹,检测mTOR敲减效果及其下游基因c-myc、周期蛋白D1(cyclinD1)表达水平及4E-BP1、S6K1磷酸化水平的变化。结果:RT-PCR和Western印迹结果显示,构建的pLenti-H1/mTOR siRNA能有效抑制mTOR基因的表达,敲低了mTOR蛋白水平,且沉默mTOR后其下游基因c-myc、CyclinD1的表达水平及4E-BP1、S6K1磷酸化水平降低。结论:构建了慢病毒介导RNA干扰mTOR表达载体,为进一步研究mTOR通路奠定了实验基础。

Construction and Preliminary Function Assay of a Lentivirus-Medi-ated mTOR Knock-Down Hepatoma Cell Line HepG2

Objective： To establish an efficient and stable method for mammalian target of rapamycin（mTOR） small interfering RNA（siRNA） delivery into hepatoma cell line HepG2 and assay its function preliminarily. Meth-ods： Two different pLenti-H1/mTOR siRNA were constructed, and they were transfected into 293T cells together with three viral packaging vectors to produce lentivirus particles. After HepG2 cells were infected with the harvest- ed viruses and experienced screening for 2 weeks with puromycin, they were collected to examine expression and function of roTOR by Western blot and quantitive RT-PCR（qPCR） analysis. Results： Western blot and qPCR showed that pLenti-H1/mTOR siRNA could suppress the roTOR gene expression. Suppression of mTOR could mark-edly down-regulate the phosphorylation level of its downstream targets such as 4E-BP1 and S6K1, and expression level of c-myc and cyclinD! genes. Conclusion： The lentivirus-mediated mTOR siRNA were obtained, which lays the foundation for further research on mTOR signaling pathway.