Mcl-1短发夹RNA在Raw264.7细胞内特异性的筛选

Specificity of screening of short hairpin RNA targeting Mcl-1 gene in Raw264.7 cells

目的:将Mcl-1shRNA转染到Raw264.7细胞内,针对shRNA对小鼠Raw264.7巨噬细胞系中Mcl-1表达的影响,筛选出沉默Mcl-1基因效果最明显的特异性shRNA真核表达质粒。方法:将特异性shRNA经脂质体介导转染小鼠巨噬细胞系Raw264.7;半定量RT-PCR和Western blot分别检测转染24、48 h后Mcl-1 mRNA水平变化和Mcl-1蛋白表达情况,分析对应不同位点的三对特异性shRNA片段对Mcl-1的沉默效果。结果:特异性shRNA片段在24、48 h均能有效降低Mcl-1 mRNA和蛋白水平,沉默效率高于正常组、脂质体组和阴性对照组,差异具有统计学意义(P<0.05);对应不同位点的三对shRNA真核表达质粒,其中Mcl-1 shRNA3对Mcl-1 mRNA和蛋白的抑制作用均最强。结论:RNA干扰技术可有效下调小鼠Raw264.7巨噬细胞系中Mcl-1 mRNA水平,明显下调Mcl-1蛋白表达。成功筛选出了沉默Mcl-1基因效果最明显的特异性shRNA真核表达质粒。

Objective：To transfect Mcl-lshRNA into Raw264. 7 cells, and screen out specific shRNA eukaryotic expression plasmids with the most significant effect of silent Mcl-1 gene to figure out the effect of shRNA on Mcl-1 expression in murine macrophage cell line Raw264. 7. Methods： Specific shRNA was transfected into murine macrophage cell line Raw264. 7 via lipofectamine. Semi-quantitative RT-PCR and Western blot were respectively employed to test the changes in Mcl-1 mRNA level and Mcl-1 protein expressions 24 h and 48 h after the transfection, and the silencing effects of the three pairs of specific shRNA fragments corresponding to different sites were analyzed. Results： Specific shRNA fragments at 24 h and 48 h could effectively reduce Mcl-1 mRNA and protein level, with higher silencing effects than those of the normal group, the lipofectamine group and the negative control group. There were statistically significant differences among them （P〈0. 05）. Among the three pairs of specific shRNA fragments corre- sponding to different sites, Mcl-1 shRNA3 showed the most significant inhibiting effect on Mcl-1 mRNA and proteins. Conclusion： RNA interference can downregulate the level of Mcl-1 mRNA in murine macrophage cell line Raw264. 7 and greatly downregulate the expression of Mcl-1 protein. Specific shRNA eukaryotic expression plasmids with the most significant effect of silent Mcl-1 gene have been screened out successfully.