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Western blot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OPG mRNA和蛋白表达均较对照差异有统计学意义(P＜0.05),RNAi对OPG mRNA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 that expresses 3 shRNAs (short hairpin RNAs) targeting osteoprotegerin (OPG) gene and to investigate the inhibitory effect of RNA interference (RNAi) on osteoprotegerin gene expression in MDAMB-231 breast cancer cell line.

Methods

Three sites targeting osteoprotegerin gene were selected. Accordingly,three pairs of single stranded DNAs were designed,synthesized and annealed to double stranded DNAs. The double stranded DNAs were cloned into pGenesil-1.1,pGenesil-1. 2 and pGenesil-1. 3,respectively. After repeated enzyme digestion and ligation,the construction of recombinant plasmid pGenesil-1. 1-1. 2-1. 3-shRNA1-shRNA2-shRNA3 was completed. After identification of restriction endonuclease and sequencing,the recombinant plasmid was transfected into the MDA-MB-231 cells. After G418 screening,the stably transfected cells were identified with RT-PCR and Western blot. Statistical analysis was performed using One-way ANOVA and LSD methods.

Results

The eukaryotic expression plasmid pGenesil-1. 1-1. 2-1. 3-shRNA1-shRNA2-shRNA3 was constructed successfully. Compared with the control group,OPG mRNA and protein levels of MDA-MB-231 cells stably transfected with pGenesil-1. 1-1. 2-1. 3-shRNA1-shRNA2-shRNA3 were lower,with a statistically significant difference (P ＜0. 05). The inhibition rates of OPG mRNA and protein were 91% and 73%,respectively.

Conclusion

The eukaryotic expression plasmid that expresses 3 shRNAs targeting osteoprotegerin gene was constructed successfully,and the osteoprotegerin gene expression in MDA-MB-231 cells was inhibited by RNA interference in our study. Our study result may provide a basis for related experimental studies further exploring the role of OPG expression by tumor cell itself in the development and progression of breast cancer bone metastasis.