RNA干涉抑制NIH3T3细胞中外源PC-1基因表达

RNA interference suppresses expression of extraneous PC-1 gene in NIH3T3 cells

目的:获得能有效抑制PC 1基因表达的RNAi靶标,用RNAi技术研究PC- 1基因表达在前列腺癌中的作用。方法:用DNA重组技术构建了可表达短发夹RNA的重组质粒,将重组质粒与表达PC-1- EGFP融合蛋白的质粒共转染NIH3T3细胞,通过荧光显微镜观察绿色荧光蛋白表达差异,从 5个候选靶标中初筛出最合适的RNAi靶标,再通过RT- PCR和Western印迹从mRNA水平和蛋白质水平检测该RNAi靶标抑制PC- 1基因表达的效果。结果:利用RNA干涉有效靶点抑制了NIH3T3细胞中外源PC -1基因表达。结论:这一策略适合大量筛选RNAi有效靶标序列,其结果为研究RNAi技术降低PC 1基因的表达对前列腺癌细胞的影响奠定了基础。

Objective: To study the correlation between PC-1 gene and prostate cancer by RNA interference technology, the most appropriate RNA interference target to PC-1 gene needs to be selected from candidates. Methods:Recombinant plasmids expressing short hairpin RNA targeting PC-1 mRNA were constructed using DNA recombinant technology and transfected into NIH3T3 cells with PC-1-EGFP plasmid respectively. The most effective RNA interferencing construct was selected from 5 candidates by observing the expression of EGFP using fluorescence microscope 48 hours after transfection. The effect of RNA interference was further confirmed by reverse transcriptase polymerase chain reaction and Western-blotting analysis. Results:PC-1 gene expression was surpressed by the most effective RNAi target. Conclusion:This strategy is adapted to select the most effective target from many candidates. The work provides basis for researhing the impact of suppression of the endogenous PC-1 gene expression on the biological behavior of prostate cancer cell.