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目的研究短发夹状RNA(shRNA)干扰慢病毒表达载体对小鼠胶质瘤GL261细胞系中乏氧诱导因子-1α(HIF-1α)基因的沉默效应。方法构建针对小鼠胶质瘤GL261细胞HIF-1αmRNA的不同干扰靶点的4个shRNA表达载体,筛选出HIF-1α基因的RNAi有效靶序列,进一步合成靶序列的Oligo DNA,构建pLenti6.3-shR-NA3慢病毒载体感染靶细胞GL261,获得稳定沉默HIF-1α细胞株,利用实时定量PCR和Western blot方法检测稳定细胞株HIF-1α的沉默效应。结果成功构建了具有HIF-1α沉默效应的慢病毒干扰载体,通过倍比稀释测定干涉病毒滴度为1.15×108TU/mL。实时定量PCR和Western blot实验均证实慢病毒转染后,GL261细胞株中HIF-1α表达水平明显降低。结论针对HIF-1α基因不同位点的不同shRNA具有干扰效率的差异。特异性的shRNA可稳定地介导HIF-1α基因沉默。
Objective To investigate the silencing effect of short hairpin RNA (shRNA) on hypoxia inducible factor-1α (HIF-1α) gene in glioma GL261 cell line by lentivirus vector. Methods Four shRNA expression vectors targeting different targets of HIF-1αmRNA in murine glioma GL261 cells were constructed. The RNAi effective target sequence of HIF-1α gene was screened out and Oligo DNA of target sequence was further synthesized to construct pLenti6.3- shR-NA3 lentiviral vector was used to infect target cell GL261 to obtain a stable silencing HIF-1α cell line. The silencing effect of stable cell line HIF-1α was detected by real-time PCR and Western blot. Results The lentiviral vector with the silencing effect of HIF-1α was successfully constructed and the titer of interfering virus was 1.15 × 108TU / mL. Real-time PCR and Western blot confirmed that the expression of HIF-1α in GL261 cells was significantly decreased after transfection with lentivirus. Conclusion Different shRNAs targeting different sites of HIF-1α gene have different interference efficiency. Specific shRNAs can stably mediate HIF-1α gene silencing.