CAJ | 학술논문

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Lipofectamine RNAiMAX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 (100 nmol/L) 48 h；第2组细胞先转染inhibitor(100 nmol/L) 24 h,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real time RT-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Lipofectamine 2000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(luciferase)检测荧光素酶的表达变化.应用SPSS 13.0统计软件,采取单因素方差分析或t检验进行数据统计分析.结果 THP-1细胞转染miR-29a的mimic48 h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,24 h后细胞凋亡较对照组增加；THP-1细胞先转染inhibitor 24 h后,再用LPS诱导24 h,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.
목적 탐토microRNA-29a (miR-29a)대인단핵세포주THP-1조망적작용급기궤제.방법 체외배양인단핵세포주THP-1화인배신세포주293T,합성인miR-29a적의사물(mimic)화억제제(inhibitor).용지질체Lipofectamine RNAiMAX전염miR-29a적mimic혹inhibitor진입THP-1세포,분조처리후수집세포표본.제1조세포전염mimic (100 nmol/L) 48 h；제2조세포선전염inhibitor(100 nmol/L) 24 h,재용지다당(LPS)유도24h,분별용류식세포의방법검측량조세포조망,용real time RT-PCR방법검측항조망기인Bcl-2화Mcl-1적표체변화.구건Bcl-2화Mcl-1적형광소매보고기인재체,용지질체Lipofectamine 2000전염293T세포(DNA질립화miRNA편단공전염),쌍형광소매보고기인계통(luciferase)검측형광소매적표체변화.응용SPSS 13.0통계연건,채취단인소방차분석혹t검험진행수거통계분석.결과 THP-1세포전염miR-29a적mimic48 h후,세포조망교대조조증가(17.38％증가지42.06％)；단독용LPS유도THP-1세포,24 h후세포조망교대조조증가；THP-1세포선전염inhibitor 24 h후,재용LPS유도24 h,세포조망교단독용LPS유도조감소(유51.50％강지38.09％)；THP-1세포전염miR-29a적mimic후,항조망기인Bcl-2화Mcl-1적표체수평강저명현(P＜0.05).령외,Luciferase검측결과현시,재293T세포중,쌍형광보고계통현시miR-29a가특이억제대유Bcl-2화Mcl-13'UTR상야생형식별원건적보고기인표체(P＜0.05).결론 상조miR-29a적표체수평능촉진THP-1세포발생조망,하조miR-29a적표체수평칙능억제LPS유도적THP-1세포조망,miR-29a조공THP-1적조망수평시통과파향우량개항조망기인Bcl-2화Mcl-1실현적,제시miR-29a재조공면역세포적조망과정중구유중요적작용.
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 cotransfected 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.