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To investigate the effects of miR-146a on the immune inflammation of the megakaryocyte/platelet.
An in vitro megakaryocytic maturation and platelet formation model,as measured by CD41 and CD61 through flow cytometry,has been established by using K562 cells treated with PMA induction. MiR-146a mimic, miR-146a inhibitor,mimic NC and inhibitor NC were respectively transfected platelets-producing megakaryocytes.48-72 hours after thansfection,the expression of IL-6 and TNF-a were evaluated by ELISA,then RT-PCR and western blot were used to evaluate TLR4 and NF-KB.In addition,the effect of miR-146a on the potential target gene IRAK1 was detected by luciferase activity assay.And RT-PCR and western blot was used to determine the effect of miR-146a on IRAK1.
The expression of IL-6,TNF-a and NF-KB was suppressed by increased levels of miR-146a and increased by the reduction of miR-146a. And TLR4 expression in megakaryocytes/platelets was increased by upregulated miR-146a and decreased by down-regulated miR-146a.Luciferase activity assay suggest that miR-146a directly suppresses IRAK1 expression by targeting the 3′UTR of IRAK1 mRNA.Mir-146a showed an inability to alter IRAK1 expression at mRNA level in megakaryocytes/platelets,however it demonstrated an ability to down-regulate IRAK1 protein expression.
Our research was first to demonstrated that miR-146a can affects the platelet immune activation via TLR4/NF-KB signal pathway,which imply that miR-146a might be able to act as novel tools for regulation of platelet immune inflammation.