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目的构建针对骨保护素(OPG)基因的一个载体编码三条短发夹RNA(shRNA)的真核表达质粒,观察其对MDA-MB-231乳腺癌细胞株OPG表达的抑制作用。方法选择3个针对OPG基因的RNA干扰(RNAi)位点,分别设计合成3对编码相应shRNA的DNA单链,每对单链连接形成双链后分别与线性化载体pGenesil-1.1、pGenesil-1.2、pGenesil-1.3连接形成pGenesil-1.1-shRNA1、pGenesil-1.2-shRNA2、pGenesil-1.3-shRNA3,对以上重组载体反复酶切连接,构建成重组质pGenesil-1.1-1.2-1.3-shRNA1-shRNA2-shRNA3,酶切鉴定和测序无误后,将该质粒转染MDA-MB-231细胞,以G418加压筛选,对转染细胞行单克隆化,稳定转染细胞行RT-PCR和Westernblot检测,确定其对OPG基因表达抑制作用。统计分析采用单因素方差分析和SLD分析法。结果成功构建了pGenesil-1.1-1.2-1.3-shRNA1-shRNA2-shRNA3重组质粒;以该质粒稳定转染MDA-MB-231细胞后其OPGmRNA和蛋白表达均较对照差异有统计学意义(P<0.05),RNAi对OPGmRNA和蛋白的表达抑制率分别为91%和73%。结论本研究构建了针对OPG的一个载体编码3条shRNA的真核表达质粒,通过RNAi抑制了MDA-MB-231细胞OPG基因表达,为进一步深入探讨肿瘤细胞自身OPG表达在乳腺癌骨转移发生发展中的作用提供了相关实验基础。
Objective To construct a eukaryotic expression plasmid encoding three short hairpin RNA (shRNA) targeting osteoprotegerin (OPG) gene and observe its inhibitory effect on OPG expression in MDA-MB-231 breast cancer cell line. Methods Three RNA interference (RNAi) sites targeting OPG gene were designed and synthesized. Three pairs of DNA single strands encoding the corresponding shRNAs were designed and synthesized. Each pair of single strands was double-stranded and then ligated with the linearized vectors pGenesil-1.1, pGenesil-1.2 , PGenesil-1.3 was ligated to form pGenesil-1.1-shRNA1, pGenesil-1.2-shRNA2 and pGenesil-1.3-shRNA3, and the recombinant plasmid pGenesil-1.1-1.2-1.3-shRNA1-shRNA2-shRNA3 After restriction enzyme digestion and sequencing, the plasmid was transfected into MDA-MB-231 cells and treated with G418 under pressure. The transfected cells were single-cloned and stably transfected cells were detected by RT-PCR and Western blot. Inhibition of OPG gene expression. Statistical analysis using one-way analysis of variance and SLD analysis. Results The recombinant plasmid pGenesil-1.1-1.2-1.3-shRNA1-shRNA2-shRNA3 was successfully constructed. The expression of OPG mRNA and protein in MDA-MB-231 cells stably transfected with this plasmid was significantly lower than that of the control (P <0.05 ), RNAi on OPG mRNA and protein expression inhibition rates were 91% and 73%. Conclusion This study constructed a vector targeting OPG eukaryotic expression vector of three shRNA shRNA RNAi inhibited OPG gene expression in MDA-MB-231 cells for further in-depth study of tumor cells own OPG expression in the development of breast cancer bone metastasis The role provided in the experimental basis.