The Impact Of MiR-146a On The Immune Inflammation Of Platelets

Objective:To investigate whether miR-146 a regulate the immune inflammation of the megakaryocyte/platelet,and to discuss the effects of miR-146 a on immune inflamm-ation of the megakaryocyte/platelet.Methods:(1) K562 cells were cultured in vitro.PMA(Phorbol-12-myristate-13-acetate) was used to induce k562 cells differentiate into megakaryocytes which have the function of producing platelets.(2) After k562 cells were differentiate into megakaryocytes/platelets, megakar-yocytes / platelets were transfected.Then,the The following experiments were divided into the following 5 groups:(1) Control group:without any intervention to the megakaryocytes/platelets;(2) miR-146 a mimic group:mi R-146 a mimic was transfected into the megakary-ocytes/platelets;(3) miR-146 a inhibitor group: miR-146 a inhibitor was transfected into the megakaryocytes/platelets;(4) miR-146 a mimic negative control group: miR-146 a mimic negative control was transfected into the megakaryocytes/platelets;(5) miR-146 a inhibitor negative control group: miR-146 a inhibitor negative control was transfected into the megakaryocytes/platelets;(3) The mRNA levels of miR-146 a was measured by real-time PCR, the concentration of IL-6 and TNF-a of cell cultural supernatants were measured by ELISA method,the mRNA levels of NF-KB was measured by RT-PCR,the protein levels of TLR4?NF-KB were measured by Western blotting.(4) Using mi RNA target prediction software tools such as TargetScan,PicTar and miRanda to predict the potential target gene of miR-146 a.Ultimately,we chose IRAK1 as the potential target gene of miR-146 a.(5) To create wide type luciferase reporter vector Pmir-Report-WT-IRAK-1-3'UTR,and mutant(MUT) luciferase reporter vector Pmir-Report-MUT-IRAK-1-3'UTR. These two reporter vectors were cotransfected with miR-146 a mimic or miR-146 a mimic negative control(mimic-nc)into megakaryocytes/platelets, and Dual Luciferase Reporter Assay System was used to detect the luminescent signal.(6) To directly detect the miR-146 a suppression validity of the target gene, megakaryocytes/platelets were transfected with mi R-146 a mimic ? miR-146 a inhibitor and their negative control respectively. The protein and mRNA levels IRAK1 were measured by Western blotting and RT-PCR.Results:(1) k562 cells were successfully differentiated into megakaryocytes by inducing with PMA.(2) Compared with the miR-146 a mimic negative control group, the miR-146 a mimic group had a significantly higher level of miR-146 a, significantly lower concentration of IL-6 and TNF-a in cell cultural supernatants,and higher protein expression of TLR4,and lower protein and mRNA levels of NF-KB.(3) Compared with the miR-146 a inhibitor negative control group, the miR-146 a inhibitor group had a significantly lower level of miR-146 a, significantly higher concentration of IL-6 and TNF-a in cell cultural supernatants,and lower protein expression of TLR4,and higher protein and mRNA levels of NF-KB.(4) By miRNA target prediction software tools, IRAK1 was predicted to be the target gene of miR-146 a.(5) Compared with cotransfection of mimic-nc with Pmir-Report-WT-IRAK-1-3'UTR group,the cotransfection of miR-146 a mimic with Pmir-Report-WT-IRAK-1-3'UTR group significantly suppressed the luciferase activity.However,cotransfection of mi R-146 a mimic or mimic-nc with Pmir-Report-MUT-IRAK-1-3'UTR didn't effect the luciferase activity.(6) Compared with the control,transfection with miR-146 a mimic downregu-lated IRAK1 protein level while transfection with miR-146 a inhibitor downregu-lated IRAK1 protein level.However,miR-146 a didn't effect the level of IRAK1 mRNA.Conclusion:(1) MiR-146 a could regulate immune inflammation of megakaryocytes/ platelets by playing an important inhibitory role in the signal path of TLR4/NF-KB.(2) MiR-146 a could effectively inhibit its target gene IRAK1,which plays an important role in the signal path of TLR4/NF-KB.Thus miR-146 a may regulate the signal path of TLR4/NF-KB through its target gene IRAK1 in platelets.