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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 investigate the effect of RNAi silencing cyclin D1 expression on the proliferation of prostate cancer cell line DU145. Methods Small interfering RNA (shRNA) targeting cyclin D1 gene was designed and synthesized and transfected into DU145 cells by liposome. The expression of cyclin D1 in DU145 cells before and after transfection was detected by RT-PCR and Western blot. The cells with the highest interference rate were selected and the viability of the cells in each group was tested by MTT cell viability assay, 3H liquid scintillation incorporation assay, clone formation assay and the like. Flow Cytometry was used to detect the changes of cell cycle phase before and after silencing cyclin D1. Western blot was used to detect the expression of p21, PCNA, Bax and bcl-2. Results cyclin D1-shRNA could effectively inhibit the expression of cyclin D1 gene and its RNA and protein expression levels were significantly decreased (P <0.05), cyclin D1-shRNA significantly inhibited cell proliferation, the difference was statistically significant (P <0.05), and made it mainly stagnated in G0 / G1 phase. Proliferation-associated negative regulatory proteins, p21 and Bax, were up-regulated, and the expression of the regulatory protein PCNA and apoptosis-associated protein bcl-2 decreased. Conclusion RNA interference of cyclin D1 gene expression significantly inhibits the viability and proliferation of prostate cancer cell DU145. The possible mechanism is that it inhibits the cell cycle progression by regulating factors such as intracellular proliferation and apoptosis.