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目的观察RNA干扰技术沉默Smad4基因表达对人前列腺癌细胞增殖能力的影响,探讨Smad4在前列腺癌恶性进展中的作用。方法用半定量RT-PCR和Western blot方法检测LNCaP、PC-3、DU145以及ARCaP亚细胞系IF11、IA8等多种不同前列腺癌细胞Smad4 mRNA及蛋白表达的情况,筛选出Smad4基因阳性表达的细胞;针对Smad4基因不同部位设计并化学合成3对不同的靶向小干扰RNA(small interfering RNA,siRNA),脂质体介导瞬时转染阳性表达Smad4的细胞,转染后48h分别用RT-PCR和Western blot检测Smad4 mRNA及蛋白表达水平的变化,用MTT法检测沉默Smad4表达对细胞增殖的影响。结果检测的5种前列腺癌细胞中仅LNCaP、PC-3、DU1453种细胞表达Smad4 mRNA及蛋白;选择PC-3细胞做siRNA转染,与阴性对照序列组(siRNA-NC)相比,转染48h后,转染SiRNA1、SiRNA2、SiRNA3的3组PC-3细胞的Smad4 mRNA表达水平均显著下降,但仅有siRNA3组Smad4蛋白表达水平显著降低。转染SiRNA3沉默Smad4基因表达后PC-3细胞的体外增殖能力显著增强(P<0.05)。结论不同前列腺癌细胞Smad4基因的表达存在差异,靶向siRNA下调Smad4的表达可增强前列腺癌细胞的增殖能力。
Objective To observe the effect of Smad4 gene silencing on the proliferation of human prostate cancer cells by RNA interference and to explore the role of Smad4 in the malignant progression of prostate cancer. Methods The expression of Smad4 mRNA and protein in LNCaP, PC-3, DU145 and ARCaP subtypes IF11, IA8 and so on were detected by semi-quantitative RT-PCR and Western blot. The Smad4 positive cells Three different pairs of small interfering RNAs (siRNAs) were designed and synthesized chemically for different sites of Smad4 gene. Liposome-mediated transient transfection of Smad4 positive cells was performed by RT-PCR Western blot was used to detect the expression of Smad4 mRNA and protein. MTT assay was used to detect the effect of Smad4 silencing on cell proliferation. Results Only LNCaP, PC-3 and DU1453 cells expressed Smad4 mRNA and protein in the five prostate cancer cell lines. PC-3 cells were selected for siRNA transfection. Compared with the negative control group (siRNA-NC) After 48h, the expression of Smad4 mRNA in three groups of PC-3 cells transfected with SiRNA1, SiRNA2 and SiRNA3 decreased significantly, but the expression level of Smad4 in siRNA3 group decreased significantly. The proliferation ability of PC-3 cells was significantly enhanced after transfection of SiRNA3 silencing Smad4 gene (P <0.05). Conclusion The expression of Smad4 in different prostate cancer cells is different. The down-regulation of Smad4 expression by siRNA can enhance the proliferation of prostate cancer cells.