南方医科大学学报

南方医科大学学报 ›› 2015, Vol. 35 ›› Issue (03): 319-.

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miR-144负性调节大鼠巨噬细胞TOLL样受体2的表达

王璇，蓝茜，刘莉，伊静，李靖，李玥，王美晨，李嘉熙，宋刘梅，李冬民   

  • 出版日期:2015-03-20 发布日期:2015-03-20

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

  • Online:2015-03-20 Published:2015-03-20

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摘要： 目的明确TOLL样受体2（TLR2）与microRNA144（miR-144）作用之间的关系。方法利用不同浓度的miR-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-qPCR检测miR-144和TLR2及其下游分子TNF-α的
表达。利用大鼠肝脏cDNA为模板，PCR获取目的片段（即含miR-144 野生及突变结合位点的TLR2 mRNA的3’UTR区）；用
SacⅠ、XbaⅠ双酶切pmirGLO报告基因载体和含miR-144野生及突变结合位点的TLR2 mRNA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miR-144 的mimics 与双荧光素酶报告基因共转染明确miR-144 与TLR2 mRNA的3’UTR 区的靶向关系。结果瞬时转染
100 nmol/L miR-144 mimics后，NR8383细胞中miR-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
miR-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 miR-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miR-144通过靶向结合TLR2 mRNA的3’UTR区负性调节TLR2及其下游促炎因子的表达。

Abstract: Objective To investigate the relationship between miR-144 and Toll-like receptor 2 (TLR2). Methods RT-qPCR 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 miR-144 or miR-144 inhibitor. The fragments of 3’UTR region of rat TLR2 mRNA including
wild or mutant miR-144 binding site obtained by PCR using rat liver cDNA were ligated to pmirGLO report gene vector
digested with SacI and XbaI to construct the recombinant vectors of pmir-TLR2-3’UTR and pmir-mutant-TLR2-3’UTR. The
miR-144 targeting TLR2 was further determined by dual luciferase reporter assay and miR-144 mimics. Results TLR2 and
TNF-α in NR8383 cells were decreased after transfection with 100 nmol/L mimic miR-144 for 24 h and increased after
transfection with 100 nmol/L miR-144 inhibitor. PCR and double-enzyme digestion with SacI and XbaI confirmed successful
insertion of the target fragments. Dual luciferase reporter assay suggested the binding of miR-144 to the 3’UTR of rat TLR2
mRNA. Conclusion miR-144 negatively regulates the expression of TLR2 and its down-stream cytokine TNF-α by targeting
TLR2 in NR8383 cells.