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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 obtain RNAi target that can effectively inhibit the expression of PC 1 gene and to study the role of PC-1 gene expression in prostate cancer by using RNAi technique. METHODS: Recombinant plasmids expressing short hairpin RNA (shRNA) were constructed by DNA recombination technique. The recombinant plasmids were co-transfected into NIH3T3 cells with the plasmid expressing PC-1-EGFP fusion protein. The fluorescence intensity of green fluorescent protein The most suitable RNAi target was screened out from the candidate targets and the effect of RNAi target inhibition of PC-1 gene expression was detected by RT-PCR and Western blotting from mRNA level and protein level. Results: The effective target of RNA interference inhibited the expression of exogenous PC-1 gene in NIH3T3 cells. Conclusion: This strategy is suitable for screening a large number of effective RNAi target sequences. The results lay a foundation for studying the effect of RNAi technology on PC1 gene expression in prostate cancer cells.