靶向ADAM17基因siRNA对前列腺癌PC-3细胞增殖能力的影响

Inhibitory effect of siRNA targeting ADAM17 on the proliferation of prostate cancer PC-3 cells

目的:探讨靶向抑制ADAM17基因对雄激素非信赖性前列腺癌PC-3细胞增殖的影响。方法:应用ADAM17 siRNA转染PC-3细胞后,通过RT-PCR、Western印迹方法分别检测ADAM17 mRNA和蛋白表达变化;MTT、BrdU掺入法检测下调ADAM17对PC-3细胞的增殖和DNA合成能力的影响;流式细胞术检测ADAM17 siR-NA对PC-3细胞细胞周期的影响;Western印迹检测下调ADAM17对PC-3细胞增殖相关基因表达的影响。结果:两对ADAM17 siRNAs均可有效地降低PC-3细胞ADAM17 mRNA和蛋白的表达;MTT结果显示与对照组(0.80±0.51)相比,两对ADAM17 siRNAs均可显著抑制细胞的生长(0.43±0.57、0.44±0.64,P均<0.05);Br-dU掺入实验显示与对照组(0.79±0.72)相比,ADAM17 siRNAs均能显著下调DNA的合成能力(0.48±0.43、0.54±0.59,P<0.05);流式细胞术结果显示,与对照组(41.38±1.53)%相比,ADAM17 siRNAs可显著增加G1期细胞数量[(61.83±2.41)%、(59.78±1.92)%,P均<0.05]、降低S期细胞数量[从(33.51±1.47)%减少到(23.64±2.56)%、(25.24±1.86)%,P均<0.05],同时伴随着cyclin D1蛋白的表达下降而p21蛋白的表达升高。结论:ADAM17 siRNA可以通过下调cyclin D1、上调p21的表达而抑制前列腺癌PC-3细胞增殖,ADAM17可能成为前列腺癌基因治疗的靶点。

Objective： To study the effect of siRNA targeting ADAM17 （ADAM17-siRNA） on the proliferation of prostate cancer PC-3 cells. Methods： After transfecting PC-3 cells with ADAM17-siRNA 1 and ADAM17-siRNA 2, we detected the expressions of ADAM17 mRNA and protein by RT- PCR and Western blotting, respectively. We measured the changes in the proliferation and DNA synthesis of PC-3 cells by MTF and bromodeoxyuridine （BrdU） incorporation assay, examined the cell cycle profile by flow cytometry, and determined the expressions of the genes associated with PC-3 cell proliferation by Western blotting. Results： Both ADAM17-siRNA 1 and 2 effectively reduced the expressions of ADAM17 mRNA and protein in the PC-3 cells. Knockdown of ADAM17with the two siRNAs significantly inhibited cell proliferation as compared with the control group （0.43 ± 0.57 and 0.44 ± 0.64 vs O. 80 ± 0.51, P 〈 0.05） and down-regulated DNA synthesis （0.48 ± 0.43 and 0. 54 ± 0. 59 vs O. 79 ± O. 72, P 〈 0.05 ）. The cell cycle profile showed that the cell population of the G1 phase was markedly higher in both the ADAM17-siRNA groups than in the control （ [ 61.83 ±2.41 ] % and [ 59.78 ± 1.92 ] % vs [ 41.38 ± 1.53 ] %, P 〈 0.05 ）, but that of the S phase remarkably lower in the former two than in the latter （ [ 23.64 ± 2.56 ] % and [ 25.24± 1.86 ] % vs [ 33.51 ± 1.47 ] %, P 〈 0.05）, with a concomitant decrease in the expression of the cell cycle protein cyclin D1 and increase in the cyclin-dependent kinase inhibitor p21. Conclusion ： ADAM17- siRNA can effectively inhibit the proliferation of PC-3 cells by up-regulating cyclin D1 and down-regulating p21 protein, and ADAM17 has a potential value in the gene therapy of prostate cancer.