MicroRNA 144 negatively regulates Toll-like receptor 2 expression in rat macrophages
10.3969/j.issn.1673-4254.2015.03.02
- VernacularTitle:miR-144负性调节大鼠巨噬细胞TOLL样受体2的表达
- Author:
Xuan WANG
1
;
Xi LAN
;
Li LIU
;
Jing YI
;
Jing LI
;
Yue LI
;
Meichen WANG
;
Jiaxi LI
;
Liumei SONG
;
Dongmin LI
Author Information
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
- Keywords:
miR-144;
Toll-like receptor 2;
NR8383 cells;
macrophage;
PmirGLO report gene carrier
- From:
Journal of Southern Medical University
2015;(3):319-325
- CountryChina
- Language:Chinese
-
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-αin 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-αby targeting TLR2 in NR8383 cells.
