MicroRNA 144 negatively regulates Toll-like receptor 2 expression in rat macrophages / 南方医科大学学报

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the relationship between miR-144 and Toll-like receptor 2 (TLR2).</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>miR-144 negatively regulates the expression of TLR2 and its down-stream cytokine TNF-α by targeting TLR2 in NR8383 cells.</p>