沉默p65基因对人乳腺癌细胞MDA-MB-231细胞周期的影响

目的研究miRNA(microRNA)沉默p65基因后对人乳腺癌细胞株MDA-MB-231细胞周期分布的影响。方法用pcDNATM6.2-GW/EmGFP-miR载体构建p65 miRNA表达质粒，转染MDA-MB-231细胞，用RT-PCR检测转染前后各组细胞中p65 mRNA表达变化；凝胶电泳迁移率改变分析(electrophoretic mobility shift assay, EMSA)实验研究转染前后细胞中NF-κB结合活性的变化；流式细胞术(Flow cytometry, FCM)观察转染前后细胞周期分布的变化；Westernblot法检测转染前后细胞周期相关因子cyclinD1、CDK4、p21蛋白表达的变化。结果p65 miRNA质粒明显下调MDA-MB-231细胞中p65 mRNA的表达水平，并显著抑制细胞中NF-κB的结合活性(P<0.05)。FCM结果显示，转染组细胞G₀/G₁期细胞比例显著增加，S、G₂/M期细胞比例明显下降(P<0.05)；Western blot分析结果显示，沉默p65基因后细胞中cyclinD1蛋白表达下调，p21蛋白表达增加。结论p65 miRNA可以显著降低p65 mRNA的表达，诱导乳腺癌细胞发生G₀/G₁期阻滞，可能是通过降低cyclinD1蛋白，上调p21蛋白表达而实现的。

Effects of Silencing p65 Gene on Cell Cycle Distribution of Human Breast Cancer MDA-MB-231 Cells

ObjectiveTo study the effects of p65 microRNA (miRNA) on cell cycle distribution of human breast cancer cell line MDA-MB-231. Methodsp65-targeted miRNA was designed and transfected into MDA -MB-231 cells via lipofecta mineTM2000 mediation. The level of p65 mRNA expression was detected by RT-PCR. NF-κB binding activity of MDA-MB-231 cells was measured by electrophoresis mobility shift assay (EMSA). Cell cycle distribution was further detected by flow cytometry (FCM).Expression of cyclinD1, CDK4 and p21 proteins were determined by Western blot after transfection. Resultsp65 miRNA expression plasmid was constructed successfully and led to decrease expression level of p65 mRNA dramatically in MDA-MB-231 cells. EMSA assay showed that NF-κB binding activity of MDA-MB-231 cells was inhibited significantly, and lower than that of miR-neg and blank control group (P<0.05). 48 h after transfection, p65 miRNA increased the cell number in G0/G1 phase and decreased the cell proportion in S and G2/M phase (P<0.05). Western blot analized results furtherly confirmed that cyclinD1 protein was inhibited in p65 miRNA-transfected cells, while p21 protein increased after transfection. ConclusionSilencing p65 gene by miRNA was able to arrest MDA-MB-231 cells phase at G0/G1 phase, which might be mediated by downregulation of cyclinD1 and upregulation of p21.