慢病毒介导RNA干扰Clusterin基因表达载体的构建及其在肾癌786-O与ACHN细胞系中的表达

目的:应用慢病毒介导的RNA干扰技术,建立Clusterin(CLU)基因稳定干扰的人肾癌细胞系,为CLU基因的功能研究奠定基础.方法:以人CLU mRNA编码序列作为干扰靶点,与pGCSIL-GFP载体连接真核表达载体.另外构建不针对任何已知mRNA的阴性对照shRNA表达质粒.利用包装细胞293T获得重组慢病毒,分别感染人肾癌786-O细胞株及ACHN细胞株.应用Real time-PCR及Western blot检测不同组别CLU表达的变化.结果:成功构建CLU shRNA慢病毒载体CLU-RNAi-LV并获得相应的慢病毒,病毒悬液滴度＞4×1011TU/L.Real Time-PCR、Western blot结果显示干扰组CLU mRNA及蛋白表达水平较对照组显著降低.结论:慢病毒介导的CLU基因干扰能稳定有效地表达于肾癌细胞,筛选出能稳定干扰CLU基因表达的siRNA序列和肾癌786-O细胞株.

Construction of a Lentivirus-Mediated Vector for Clusterin Gene Silencing and Its Stable Expression in Human Renal Cell Cancer Line 786-O or ACHN

Objective: To construct a lentivirus-mediated vector for RNA interference(RNAi) of clusterin(CLU) and to establish a human renal cancer cell line with CLU gene interference.Methods: Lentiviral vectors for different short hairpin RNAs targeting the coding region of human CLU mRNA(CLU-RNAi-LV) and another control vector containing a nonsense sequence were constructed.The recombinant lentiviral vectors were harvested from 293T cells and were used to transfect human renal cancer cell lines 786-O and ACHN.Expressions of CLU mRNA and protein in the cells were detected by real time-PCR and Western blot respectively.Results: The lentiviral vectors CLU-RNAi-LV were constructed successfully and confirmed by DNA sequencing.CLU gene targeting via RNAi mediated by lentivirus vector had obviously inhibitory effects on expressions of CLU mRNA and protein in the human renal cancer cell line 786-O and ACHN.Compared with the control vector,CLU-RNAi-LV resulted in obviously reduced expression of CLU mRNA and protein in 786-O cell line.Conclusion: Lentivirus-mediated CLU RNAi can specifically inhibit CLU expression in human renal carcinoma cell line 786-O with CLU gene stable interference.