干扰cyclinD1表达对前列腺癌细胞系DU145增殖及相关基因表达的影响  

Effect of interfering cyclin D1 expression on the DU145 proliferation of prostate cancer cell line and the related gene expression

作　　者：叶挺宇[1] 卢湧湧[1] 潘悦[1] 谢辉[1] 

机构地区：[1]温州医科大学附属第一医院泌尿外科,浙江温州325000

出　　处：《中国卫生检验杂志》2015年第13期2071-2074,共4页Chinese Journal of Health Laboratory Technology

基　　金：浙江省自然科学基金(Y12H310019)

摘　　要：目的本研究探讨RNA干扰技术沉默cyclin D1的表达对前列腺癌细胞系DU145增殖过程的影响。方法设计并合成靶向cyclin D1基因的小分子干扰RNA质粒,并以脂质体介导转染入DU145细胞。用RT-PCR和Western blot检测转染前后DU145细胞中cyclin D1的表达水平。挑选干扰率最高的细胞,以MTT细胞活力检测、3H液闪掺入试验、克隆形成实验等检测各组细胞的存活率。然后采用流式细胞法检测沉默cyclin D1前后细胞周期相期的分布变化,Western blot检测细胞增殖相关调节基因p21、PCNA、Bax以及bcl-2的表达变化。结果 cyclin D1-shRNA能有效抑制cyclin D1基因的表达,其RNA和蛋白表达水平都显著下降,差异有统计学意义(P<0.05);cyclin D1-shRNA则显著抑制细胞增殖,差异有统计学意义(P<0.05),并使其主要停滞在G0/G1期。增殖相关负调节蛋白p21和Bax表达上调,正调节蛋白PCNA和凋亡拮抗蛋白bcl-2表达则下降。结论 RNA干扰cyclin D1基因表达明显抑制前列腺癌细胞DU145的活力及增殖能力,其机制可能是通过调节胞内增殖及凋亡等相关因子而阻碍其细胞周期进程。Objective To explore the effect of RNA interfering technology silencing cyclin D1 expression on the DU145 prolifer- ation process of prostate cancer cell line. Methods A small interfering RNA plasmid targeting cyclin D1 was designed and syn- thesized, and transfected into DU145 ceils by liposome. Cyclin D1 expression level in DU145 cells was detected by RT -PCR and Western blot before and after transfection. After selecting the cells with the highest interference rate, MTT cell activity test, 3H liquid flash incorporation test and colony forming experiment were used to detect the cell survival rate of each group, and flow cytometry was used to detect the change of cell cycle phase distribution before and after silencing cyclin D1. The expression changes of the related regulating genes of cell proliferation, p21, PCNA, Bax and bcl - 2, were detected by Western Blot. Results Cyclin D1 - shRNA can effectively inhibit the expression of cyclin D1 gene, the RNA and protein expression levels decreased significantly, the difference was statistically significant （ P 〈 0.05 ） ; cyclin D1 - shRNA inhibited cell proliferation, the difference was statistically significant （P 〈 0.05 ） ; and made it mainly arrested in G0/G1 phase. The expression of p21 and Bax were up - regulated, and the expression of PCNA and apoptosis protein antagonist bcl - 2 decreased. Conclusion RNA interfeting cyclin D1 expression can obviously inhibit DU145 prostate cancer cells viability and proliferation ability, and block the cell cycle progression. The mechanism may be the regulation of cell proliferation and apoptosis related factors.

关 键 词：前列腺癌 细胞周期蛋白D1 RNA干扰 增殖 

分 类 号：R737.25[医药卫生—肿瘤]