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目的构建Smad泛素调节因子1(Smurf1)基因的RNA干扰(RNAi)慢病毒表达载体,观察其转染骨肉瘤U20S细胞后对Smurf1基因表达的抑制作用,为后续研究奠定基础。方法构建Smurf1基因过表达质粒,同时针对Smurf1基因序列设计合成4条RNAi片段并构建RNAi慢病毒载体。过表达质粒和RNAi质粒共转染293T细胞后应用Western印迹法进行外源筛靶,筛靶出成功的干扰质粒PSC2939。用慢病毒包装PSC2939后感染U20S细胞,进行内源验证,荧光定量PCR和Western印迹检测U20S细胞中Smurf1mRNA及蛋白的表达。结果经PCR及测序证实,Smurf1基因过表达载体及4种干扰载体均成功构建;Western印迹外源筛靶显示,PSC2939干扰载体能有效敲减目的基因的表达;慢病毒包装后的PSC2939干扰载体能有效抑制U20S细胞中Smurf1蛋白的表达,对Smurf1mRNA的抑制率达到60%以上。结论成功构建可供感染的Smurf1基因RNAi慢病毒载体,为进一步研究Smurf1基因在骨肉瘤细胞中的作用提供了有效的实验工具。
Objective To construct RNA interference (RNAi) lentiviral expression vector of Smurf1 gene and observe its inhibitory effect on Smurf1 gene expression in transfected osteosarcoma U20S cells, which will lay the foundation for further study. Methods Smurf1 gene overexpression plasmid was constructed. At the same time, four RNAi fragments were designed and synthesized based on Smurf1 gene sequence and RNAi lentiviral vector was constructed. 293T cells were cotransfected with overexpression plasmid and RNAi plasmid, then Western blotting was used to screen the target cells and the target plasmid PSC2939 was screened out. U20S cells were infected with PSC2939 after lentivirus packaging, and the expression of Smurf1 mRNA and protein in U20S cells was detected by fluorescence quantitative PCR and Western blot. The results of PCR and sequencing confirmed that the Smurf1 gene overexpression vector and four kinds of interference vectors were successfully constructed; Western blot screening showed that PSC2939 interference vector can effectively knockdown the expression of the target gene; lentivirus packaging PSC2939 interference vector Effectively inhibit the expression of Smurf1 protein in U20S cells, and inhibit the expression of Smurf1 mRNA to over 60%. Conclusion The RNAi lentiviral vector of Smurfl gene was successfully constructed, which provided an effective experimental tool for further study on the role of Smurfl gene in osteosarcoma cells.