MicroRNA-29a在脂多糖诱导人单核细胞凋亡中的作用和机制  

MicroRNA-29a regulates apoptosis induced by lipopolysaccharide in THP-1 cells

作　　者：熊旭明[1] 张振辉[1] 江子欣[1] 陈伟燕[1] 杨其霖[1] 刘卫江[1] 

机构地区：[1]广州医学院第二附属医院重症医学科,广州510260

出　　处：《中华急诊医学杂志》2013年第1期40-45,共6页Chinese Journal of Emergency Medicine

基　　金：广东省自然科学基金（10151018201000018）;广州市医药卫生科技项目（201102A213020）

摘　　要：目的探讨microRNA-29a（miR-29a）对人单核细胞株THP-1凋亡的作用及其机制。方法体外培养人单核细胞株THP-1和人胚肾细胞株293T，合成人miR-29a的拟似物（mimic）和抑制剂（inhibitor）。用脂质体LipofectamineRNAiMAX转染miR-29a的mimic或inhibitor进入THP-1细胞，分组处理后收集细胞标本。第1组细胞转染mimic（100nmol／L）48h；第2组细胞先转染inhibitor（100nmolfL）24h，再用脂多糖（LPS）诱导24h，分别用流式细胞仪方法检测两组细胞凋亡，用realtimeRT—PCR方法检测抗凋亡基因Bcl-2和Mcl-1的表达变化。构建Bcl-2和Mcl-1的荧光素酶报告基因载体，用脂质体Lipofectamine2000转染293T细胞（DNA质粒和miRNA片段共转染），双荧光素酶报告基因系统（1uciferase）检测荧光素酶的表达变化。应用SPSS13．0统计软件，采取单因素方差分析或￡检验进行数据统计分析。结果THP-1细胞转染miR-29a的mimic48h后，细胞凋亡较对照组增加（17．38％增加至42．06％）；单独用LPS诱导THP-1细胞，24h后细胞凋亡较对照组增加；THP-1细胞先转染inhibitor24h后，再用LPS诱导24h，细胞凋亡较单独用LPS诱导组减少（由51．50％降至38．09％）；THP·1细胞转染miR-29a的mimic后，抗凋亡基因Bcl-2和Mcl-1的表达水平降低明显（P〈0．05）。另外，Luciferase检测结果显示，在293T细胞中，双萤光报告系统显示miR-29a可特异抑制带有Bcl-2和Mcl-13’UTR上野生型识别元件的报告基因表达（P〈0．05）。结论上调miR-29a的表达水平能促进THP-1细胞发生凋亡，下调miR-29a的表达水平则能抑制LPS诱导的THP-1细胞凋亡，miR-29a调控THP-1的凋亡水平是通过靶向于两个抗凋亡基因Bcl-2和Mcl-1实现的，提示miR-29a在调控免疫细胞的凋亡过程中具有重要的作用。Objective To investigate the effects of microRNA-29a （miR-29a） on lipopolysaccharide （LPS） -induced apoptosis in human monocytes THP-1 cells in order to understand the molecular mechanisms. Methods Human monocytes THP-1 cell line were exposed to LPS after transfected with miR- 29a inhibitors （100 nmol/L） or just transfected with miR-29a mimic （100 nmol/L） by lipofectamine RNAiMAX. Flow cytometry （FCM） was used to detect the cell apoptosis. Real-time RT-PCR was employed to measure expressive levels of the gene Bcl-2 and Mcl-1. The luciferase assay was performed in HEK293T cells, which were co-transfected with plasmid DNA and miRNA by using Lipofectamine 2000. Statistical analysis carried out by using SPSS 13.0 software for One-way ANOVA and Student＇ s t test. Results Transfection with miR-29a mimics for 48 h increased apoptosis rate and significantly reduced the expressions of Bcl-2 and Mcl-1 in THP-1 cells in comparsion with the control group. The apoptosis rate also raised in THP-1 cell stimulated by LPS for 24 h followed by LPS stimulation for 24 h, the apoptosis rate was decreased in comparison with the LPS group. In addition, our luciferase assay data showed that HEK293T cells co-transfected with miR-29a mimics and Bcl-2 3＇ UTR-Wt or Mcl-1 3＇ UTR-Wt plasmid significantly reduced the luciferase activity compared with the control group. Conclusions The miR-29a may regulate apoptosis by targeting the genes Bcl-2 and Mcl-1, and miR-29a may play a pivotal role in the process of apoptosis in immune cells.

关 键 词：微小RNA 单核细胞 脂多糖 凋亡 BCL-2基因 MCL-1基因 

分 类 号：R[医药卫生]