PSMA增强子/启动子特异性驱动shRNA靶向干扰EGFP的研究

Study of short-hairpin RNA targeting EGFP driven specificly by PSMA enhancer/promoter

目的:探讨前列腺特异性膜抗原增强子、启动子(PSMAe/p)驱动shRNA转录效果,比较何种终止序列最有效。方法:分别以poly(A),minipoly(A),poly(U)为终止序列设计针对增强型绿色荧光蛋白(EGFP)的干扰序列,克隆入经SalⅠ和BamHⅠ双酶切的pPSMAe/p载体上。然后通过脂质体介导,将各重组干扰质粒与pEGFP-C1质粒共转染PC-3和LNCaP细胞,采用荧光显微镜,流式细胞仪,逆转录聚合酶链反应(RT-PCR)和Western blot检测EGFP在各组细胞中的干扰效果。结果:重组质粒中成功插入了目的基因片段;荧光显微镜和流式测定结果显示各干扰质粒与pEGFP-C1质粒共转染LNCaP后,各干扰组荧光表达均有不同程度减少,与空载体组相比均有统计学差异(P<0.01),pPSMAe/p-shEGFP-poly(A)组减少与pPSMAe/p-shEGFP-minipoly(A)组相比有统计学差异(P<0.05),与pPSMAe/p-shEGFP-poly(U)组相比有显著统计学差异(P<0.01)。各干扰载体与pEGFP-C1质粒共转染PC-3后各组荧光表达无统计学差异(P>0.05),EGFP mRNA和蛋白表达水平无明显差异;LNCaP细胞各干扰组中EGFP mRNA和蛋白表达与空载体对照组相比均有不同程度下降,以pPSMAe/p-shEGFP-poly(A)组最为明显。结论:成功构建了针对EGFP的RNA干扰表达载体pPSMAe/p-shEGFP-poly(A)、pPSMAe/p-shEGFP-minipoly(A)和pPSMAe/p-shEGFP-poly(U);PSMAe/p可特异性驱动shRNA转录;poly(A)终止信号终止转录最有效。

Objective： To study the effects of prostate specific membrane antigen promoter and enhancer （PSMAe/p） in driving short hairpin RNA （shRNA） transcription and explore which terminator is most effective. Methods：The designed interference sequence targeting EGFP using poly （A）,minipoly（A）, poly（U） termination signals respectively were cloned into the PSMAe/p vector ,which was ligated after Sal Ⅰ and BamH Ⅰ digestion.Than the recombinant plasmids and pEGFP-C1 plasmid were co-transfected into PC-3 and LNCaP cells using Lipofectamine2000.The suppression effect of EGFP was assayed by fluorescence microscope and flow cytometry. mRNA and protein levels of EGFP were detected by RT-PCR and Western blot in each group cells. Results： The recombinant plasmids were constructed successfully . The expression of EGFP was decreased in the LNCaP cells which were co-transfected pEGFP-C1 and each in- terfering plasmids. In comparison with that of control group, there were significant difference （P〈0.01）.The levels of EGFP in pPSMAe/p-shEGFP-poly （A） group decreased more obviously than that of pPSMAe/p- shEGFP-minipoly（A） group and pPSMAe/p-shEGFP-poly（U） group. In the PC-3 cells,the levels of EGFP had no difference （P〉0.05）. Conclusion： pPSMAe/p-shEGFP expression vectors having different termination signals targeting EGFP are successfully constructed.PSMAe/p can drive shRNA transcription specificly.Poly（A） signal is most effective in three termination signals.