摘要
目的:探讨microRNA-29a(miR-29a)对大鼠心肌细胞(CM细胞)B细胞白血病/淋巴瘤-2(Bcl-2)和髓样细胞白血病-1(Mcl-1)表达的调控机制。方法:体外培养新生Sprague-Dawley大鼠CM细胞和人胚肾细胞株293T。合成大鼠miR-29a的拟似物(mimic)和抑制剂(inhibitor)。用脂质体Lipofectamine RNAiMAX转染miR-29a mimic进入CM细胞,转染48 h后分别用实时荧光定量PCR和Western blotting检测Bcl-2和Mcl-1mRNA和蛋白的表达变化。构建Bcl-2和Mcl-1的萤光素酶报告基因载体(DNA质粒),转染293T细胞(DNA质粒和miRNA共转染)和CM细胞(miRNA和DNA质粒先后转染),双萤光素酶报告基因系统检测萤光素酶的表达变化。结果:CM细胞转染miR-29a mimic 48 h后,Bcl-2和Mcl-1 mRNA和蛋白的水平均下调(P<0.05)。双萤光素酶报告基因系统显示,在293T细胞中,miR-29a可特异抑制带有bcl-2和mcl-1 3’UTR上野生型识别元件的报告基因表达(P<0.05),在CM细胞中,抑制内源性的miR-29a水平能促进bcl-2和mcl-1 3’UTR上野生型识别元件的报告基因表达。结论:抗凋亡基因bcl-2和mcl-1是miR-29a的靶基因。miR-29a可能通过作用于Bcl-2和Mcl-1实现对心肌细胞凋亡的调控作用,具体效应仍待进一步阐明。
AIM： To investigate the regulatory mechanisms of microRNA-29a（miR-29a） on the expression of Bcl-2 and Mcl-1 in rat cardiomyocytes（CM cells）.METHODS： The CM cells were isolated from the hearts of newborn rats and transfected with miR-29a mimic（100 nmol/L） by Lipofectamine RNAiMAX.The expression of Bcl-2 and Mcl-1 at mRNA and protein levels was detected by real-time fluorescence quantitative PCR and Western blotting.The luciferase assay was performed in HEK293T cells and CM cells,which were co-transfected with plasmid DNA and miRNA using Lipofectamine 2000.RESULTS： Transfection of miR-29a mimics significantly reduced the expression levels of Bcl-2 and Mcl-1 in CM cells as compared with the control cells（P0.05）.In addition,HEK293T cells co-transfected with miR-29a mimic and Bcl-2-3＇UTR-WT or Mcl-1-3＇UTR-WT plasmid significantly reduced the luciferase activity as compared with control group（P0.05）.While CM cells transfected with miR-29a inhibitor and Bcl-2-3＇UTR-WT or Mcl-1-3＇UTR-WT plasmid in succession,the luciferase activity was increased inversely（P0.05）.CONCLUSION： miR-29a may regulate apoptosis by targeting the bcl-2 and mcl-1 genes.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2012年第11期1928-1932,共5页
Chinese Journal of Pathophysiology
基金
广东省自然科学基金资助项目(No.9151052002000006)
国家自然科学基金青年项目(No.81201453)
广州市医药卫生科技项目(No.201102A213020)
