miR-144负性调节大鼠巨噬细胞TOLL样受体2的表达  

MicroRNA 144 negatively regulates Toll-like receptor 2 expression in rat macrophages

作　　者：王璇[1,2] 蓝茜[1,2] 刘莉[1,2] 伊静[1,2] 李靖[1,2] 李玥[1,2] 王美晨[1,2,3] 李嘉熙[1,2,3] 宋刘梅[1,2,3] 李冬民[1,2] 

机构地区：[1]西安交通大学医学部基础医学院生物化学与分子生物学系,陕西西安710061 [2],陕西西安710061 [3]西安交通大学环境与疾病相关基因教育部重点实验室,陕西西安710061 [3]西安交通大学2010级临床硕士,陕西西安710061

出　　处：《南方医科大学学报》2015年第3期319-325,共7页Journal of Southern Medical University

基　　金：国家自然科学基金(81370952);国家级大学生创新训练项目(201310698055);陕西省科技计划项目(2013K21-22-03);西安交通大学本科生科研训练和实践创新基金项目(2013273及2014X15003)~~

摘　　要：目的明确TOLL样受体2(TLR2)与micro RNA144(mi R-144)作用之间的关系。方法利用不同浓度的mi R-144的模拟物(mimics)和抑制剂(inhibitor)瞬时转染大鼠巨噬细胞系NR8383细胞,RT-q PCR检测mi R-144和TLR2及其下游分子TNF-α的表达。利用大鼠肝脏c DNA为模板,PCR获取目的片段(即含mi R-144野生及突变结合位点的TLR2 m RNA的3'UTR区);用SacⅠ、XbaⅠ双酶切pmir GLO报告基因载体和含mi R-144野生及突变结合位点的TLR2 m RNA的3'UTR区,构建携带上述片段的双荧光素酶报告基因,并通过PCR、双酶切、DNA测序鉴定构建的重组质粒,即pmir-TLR2-3'UTR及pmir-mutant-TLR2-3'UTR;并用mi R-144的mimics与双荧光素酶报告基因共转染明确mi R-144与TLR2 m RNA的3'UTR区的靶向关系。结果瞬时转染100 nmol/L mi R-144 mimics后,NR8383细胞中mi R-144表达显著升高,而TLR2及其下游分子TNF-α表达显著下降;而100 nmol/L mi R-144 inhibitor作用则相反。PCR和双酶切DNA测序结果证实pmir-TLR2-3'UTR及pmir-mutant-TLR2-3'UTR重组载体构建成功;用100 nmol/L mi R-144 mimics与空载体及上述两个构建体分别共转染HEK 293T细胞后,pmir-TLR2-3'UTR转染组相对荧光素酶活性显著降低。结论 mi R-144通过靶向结合TLR2 m RNA的3'UTR区负性调节TLR2及其下游促炎因子的表达。Objective To investigate the relationship between mi R-144 and Toll-like receptor 2（TLR2）. Methods RT-q PCR was used to determine the expression of TLR2 and its downstream inflammatory cytokine TNF- α in rat macrophage cell line NR8383 transfected by a mimic mi R- 144 or mi R- 144 inhibitor. The fragments of 3＇UTR region of rat TLR2 m RNA including wild or mutant mi R- 144 binding site obtained by PCR using rat liver c DNA were ligated to pmir GLO report gene vector digested with Sac I and Xba I to construct the recombinant vectors of pmir-TLR2-3＇UTR and pmir-mutant-TLR2-3＇UTR. The mi R-144 targeting TLR2 was further determined by dual luciferase reporter assay and mi R-144 mimics. Results TLR2 and TNF-α in NR8383 cells were decreased after transfection with 100 nmol/L mimic mi R-144 for 24 h and increased after transfection with100 nmol/L mi R- 144 inhibitor. PCR and double- enzyme digestion with Sac I and Xba I confirmed successful insertion of the target fragments. Dual luciferase reporter assay suggested the binding of mi R- 144 to the 3＇UTR of rat TLR2 m RNA.Conclusion mi R-144 negatively regulates the expression of TLR2 and its down-stream cytokine TNF- α by targeting TLR2 in NR8383 cells.

关 键 词：miR-144 TOLL样受体2 NR8383细胞 巨噬细胞 pmirGLO报告基因载体 

分 类 号：R346[医药卫生—基础医学]