siRNA-survivin与GRIM-19共表达质粒的构建及其对前列腺癌DU145细胞生长的抑制作用

Construction of siRNA-survivin and GRIM-19 coexpression plasmid and its growth inhibitory effect on prostatic cancer DU145 cells

目的:构建siRNA-survivin与GRIM-19共表达质粒pGRIM-19-si-survivin并鉴定,观察pGRIM-19-si-survivin质粒对人前列腺癌DU145细胞survivin和GRIM-19 mRNA表达水平及细胞增殖能力的影响。方法:根据前期工作,利用基因重组技术构建siRNA-survivin与GRIM-19共表达质粒,将成功构建的pGRIM-19-si-survivin质粒转染人前列腺癌DU145细胞株,应用半定量RT-PCR方法检测细胞内survivin和GRIM-19 mRNA表达水平,应用MTT法观察其对细胞增殖能力的影响。结果:(1)应用酶切及质粒测序法鉴定,证明成功构建siRNA-survivin与GRIM-19共表达质粒。(2)与mock组比较,psi-survivin、pGRIM-19及pGRIM-19-si-survivin组survivin mRNA表达水平分别为0.55±0.05、0.62±0.08和0.35±0.05,差异显著(P<0.01);与psi-survivin和pGRIM-19组比较,pGRIM-19-si-survivin的抑制survivin表达作用明显增强(P<0.05);psi-survivin、pGRIM-19和pGRIM-19-si-survivin组GRIM-19表达增强,分别为mock组的1.93±0.14、2.57±0.20和4.12±0.21(P<0.01),与pGRIM-19组比较,pGRIM-19-si-survivin增强GRIM-19表达作用更明显(P<0.05)。(3)转染48 h后,与mock组比较,psi-survivin、pGRIM-19和pGRIM-19-si-survivin 3组细胞增殖率分别为58.0%±7.2%、62.1%±6.1%和50.2%±4.8%,差异显著(P<0.05);转染72 h后,与mock组比较,3组细胞增殖率分别为43.4%±4.3%、51.3%±6.7%和26.8%±7.1%,差异明显(P<0.05,P<0.01);与psi-survivin和pGRIM-19组比较,pGRIM-19-si-survivin抑制作用明显增强(P<0.05)。结论:成功构建siRNA-survivin和GRIM-19共表达质粒pGRIM-19-si-survivin;该质粒抑制survivin表达并增强GRIM-19表达,对前列腺癌DU145细胞具有协同增殖抑制效应。

AIM： To consti＇uct the recombinant plasmid that expresses siRNA - survivin and GRIM - 19 simul- taneously, and to identify the validity of the recombinant plasmid and observe its effect on expression of survivin and GRIM - 19 and proliferation ability of prostate cancer DU145 cells. METHODS ： The recombinant plasmid coexpressing siRNA - survivin and GRIM - 19 was constructed using gene cloning technique. The prostatic cancer DU145 cells were transfect- ed with the coexpression plasmid and control plasmids. Survivin and GRIM - 19 mRNA expression was detected by semi - quantitative RT- PCR. The proliferation ability affected by coexpression plasmid was measured by MTT assay. RE- SULTS： The coexpression plasmid pGRIM - 19 - si - survivin was successfully constructed according to DNA recombinant technique and identified through restriction enzyme digestion and plasmid sequencing. Compared with the mock, survivin mRNA expression levels were 0. 55±0. 05,0. 62 ±0. 08 and 0. 35 ±0. 05 in psi - survivin, pGRIM - 19 and pGRIM - 19 - si - survivin groups, respectively. Compared with psi - survivin and pGRIM - 19, pGRIM - 19 - si - survivin inhibited sttrvivin mRNA expression markedly （ P 〈 0. 05 ）, while the expression levels of GRIM - 19 mRNA were 1.93 ± 0. 14, 2. 57±0. 20 and 4. 12 ±0. 21 in psi - survivin, pGRIM - 19 and pGBIM - 19 - si - survivin groups, respectively （P 〈 0. 01 ）. Compared with pGRIM - 19 group, pGRIM -19 - si - survivin enhanced GRIM - 19 mRNA expression more obviously （P 〈0. 05 ）. After transfection for 48 h, the proliferation rates were 58.0% ± 7. 2%, 62. 1% ＋ 6. 1% and 50. 2% ±4. 8% in the 3 experiment groups compared with the mock （P 〈0. 05）. After transfection for 72 h, the proliferation rate were 43.4% ±4. 3%, 51.3% ±6. 7% and26. 8% ±7. 1% in experiment groups compared with the mock （P 〈0. 05）. Compared with psi - survivin and pGRIM - 19, pGRIM - 19 - si - survivin significantly inhibited the cell growth （ P 〈 0. 05 ）. CONCLUSION： Transfection of coexpressi0n plasmid pGRIM - 19 - si - survivin dramatically changes the mRNA expression of survivin and GRIM - 19 and inhibits the cell growth.