摘要
目的利用miRNA靶向沉默p65基因,观察其对人乳腺癌细胞MDA-MB-231增殖及凋亡的影响。方法以脂质体LipofectamineTM2000为载体,将针对特异靶点p65基因的miRNA转染入MDA-MB-231细胞。RT-PCR和Western blot法检测p65 mRNA和蛋白表达的变化;MTT法检测细胞增殖;流式细胞技术(FCM)检测细胞凋亡;Western blot法检测凋亡相关因子表达的变化。结果 RT-PCR和Western blot检测结果显示,p65miRNA转染组p65 mRNA及蛋白表达量较对照组显著下降(P<0.05);MTT检测结果显示,沉默p65基因后,细胞出现了生长抑制现象;FCM检测结果发现,p65miRNA转染组细胞凋亡率显著增加(P<0.05)。Western blot进一步检测发现,转染组细胞中caspase-3的前体蛋白条带变细,裂解片段条带加深;PARP蛋白出现裂解片段。结论 miRNA靶向沉默p65基因可抑制人乳腺癌细胞增殖,促进细胞凋亡,推测p65基因可能成为乳腺癌基因治疗的一个新靶点。
Objective To explore the effect of microRNA（miRNA）-mediated p65 gene knockdown on the proliferation and apoptosis of human breast cancer MDA-MB-231 cells.Methods p65-targeted miRNA plasmid was constructed and transfected into MDA-MB-231 cells via lipofectamineTM2000.The expression of p65 gene in the transfected cells at the mRNA and protein levels were detected by RT-PCR and Western blotting,respectively.The cell proliferation and apoptosis were measured by MTT assay and flow cytometry（FCM）,respectively.The expressions of apoptosis-related proteins were detected by Western blotting in the transfected cells.Results Compared with the negative control group,the expressions of p65 mRNA and protein in p65miRNA-trasnfected cells were significantly down-regulated（P0.05）.MTT assay showed significantly lowered viability of MDA-MB-231 cells after the transfection（P0.05）.FCM showed an increased cell apoptosis rate in p65miRNA group compared with that in the negative control group（P0.05）.Caspase-3 and PARP were both cleaved into their active forms and the expression of these active forms was increased in p65miRNA group.Conclusion The miRNA targeting p65 gene can inhibit the proliferation and induce apoptosis of breast cancer cells,and p65 gene might become a new target in gene therapy for human breast cancer.
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2011年第10期1742-1747,共6页
Journal of Southern Medical University
基金
河北省科学技术研究与发展计划项目(10276167)
河北省卫生厅医学科学研究重点课题计划项目(20110481)
