Runx2特异性siRNA筛选和功能鉴定

Selection and Functional Identification of Runx2 Specific siRNA

目的:筛选Runx2特异性siRNA,优化反应条件,抑制MC3T3-E1细胞的成骨分化,探讨异位骨化基因治疗的新思路。方法:使用Ambion公司提供的网上工具,设计5条针对小鼠Runx2的mRNA的模板,用体外转录合成法合成5条siRNA;采用RT-PCR和Western blot分别从mRNA和蛋白质两个水平,在培养的MC3T3-E1成骨前体细胞中筛选有明显抑制作用的siRNA,并优化反应条件。将筛选出的siRNA转染成骨细胞,在不同时间点检测成骨细胞相关基因I型胶原、骨桥蛋白、骨钙蛋白、骨涎蛋白mRNA的表达,采用碱性磷酸酶活性分析检测碱性磷酸酶活性的变化。结果:在合成的5条siRNA中,siRNA1657-1677对Runx2的抑制效果最明显,最佳反应条件是:浓度80nM、脂质体/siRNA为1∶1、转染时间为72小时。采用RNAi技术抑制Runx2的表达可以抑制成骨相关基因如I型胶原、碱性磷酸酶、骨桥蛋白和骨钙蛋白等的表达,阻止成骨细胞分化。结论:Runx2特异性siRNA可以抑制成骨细胞分化,可用于异位骨化基因治疗的实验研究。

Objective To Find an optimal reaction condition for Runx2 specific siRNA（small interfering RNA） for the purpose of inhibiting osteoblast differentiation. Methods The Runx2 specific siRNA was synthesized in vitro transcription and then transfected into MC3T3-E1 cells with liposome to screen out an efficacious siRNA. The efficacious siRNA was transfected into cells, and total mRNA and protein were extracted. The expression of Runx2 and osteogenic genes was assessed by RT-PCR and Western blot. The alkaline phosphatase activity analysis was investigated. Results Our results show that the runx2/Cbfal-specific siRNA inhibits the expression of Runx2 at the level of mRNA and protein. The expression levels of osteoblast maturation genes, type I collagen, osteopontin,bone sialoprotein,and osteocalcin are significantly decreased. The alkaline phosphatase activity in MC3T3-E1 cells is inhibited. Conclusion These studies show Runx2 specific siRNA inhibits differentiation in cultured MC3T3-E1 cells. It is likely that the inhibition of Runx2 by RNAi could be developed as a powerful approach to prevent or treat heterotopic ossification.