A Study On The Expression And Function Of B Cell-related MiRNAs In Immune Thrombocytopenia

Objective:To invesrigate the expression profiles of five miRNAs(miR-144-3p,miR-3162-3p,miR-320c,let-7b-5p,miR-142-3p)in peripheral blood B lymphocytes of immune thrombocytopenia(ITP)patients and healthy controls,and analyze the association between miRNA expression and ITP disease;further study the regulation mechanism of let-7b-5p about B cell function in the pathogenesis of ITP;immunization of CD61 knockout mice to construct antigen-specific ITP mouse model.Provide a novelbasis for exploring thecomplicated pathogenesis of ITP.Methods:We measured the expression of five related miRNAs in ITP peripheral blood B cells by realtime fluorescence quantitative polymerase chain reaction(qRT-PCR),and compared with healthy controls.The CD19+B cells were isolated from peripheral blood mononuclear cells(PBMCs)using immunomagnetic microbeads.PBMCs were isolated by Ficoll density gradient centrifugation.Plasma platelet autoantibody specific for GPIIb/IIIaor GPIb/IX levels were measured using the MAIPA method,and analyze the profile of miRNAs between autoantibody-positive and autoantibody-negative ITP patients.In vitro,normal human B cells were isolated,and the expression level of let-7b-5p was detected after stimulation.We screened potential target gene of let-7b-5p by bioinformatics prediction,and detected ADAM 10 by qRT-PCR.The expression of BAFF-R on the surface of CD19+B cells in ITP patients was detected by flow cytometry.Next,we analyzed the association between the relative expression of let-7b-5p and ADAM 10 relative expression in B cells of ITP patients.The CD61 knockut mouse were transfused with WT type platelets weekly.Immunization was monitored by measuring immunoglobulin G(IgG)anti-CD61 antibody production in the serum by flow cytometry.SCID mice were injected intraperitoneally with splenocyte.Platelet counts and serum platelet CD61 specific antibodies were detected weekly.Results:A total of 61 ITP patients and 31 healthy controls were measured.Compared with healthy controls,we found in ITP patients the relative expression level of miR-3162-3p(1.964 ± 2.110 vs 0.829 ± 0.952,P<0.05),and let-7b-5p(1.401 ± 0.984 vs 0.609±0.661,P<0.001),miR-142-3p(0.635 ± 0.551 vs 0.318 ± 0.354,P<0.05)were increased.In addition,no significant difference was found in the relative expression levels of miR-144-3p and miR-320.ITP Patients were divided into two groups,autoantibody-positive group(n=27)and autoantibody-negative group(n=34).Our results showed that the relative expression level of let-7b-5p was significantly increased in B cells of autoantibody-positive ITP patients compared with autoantibody-negative group(1.621 ±0.97 vs 1.048±0.696,P<0.05).No significant difference was found in the expression levels of the other four miRNAs(miR-144-3p,miR-3162-3p,miR-320c,miR-142-3p)between different autoantibody groups.In vitro,compared with the untreated control group,results showed that the relative expression levels of let-7b-5p were significantly increased in the three groups after stimulate B cell activation.Including BAFF group(74.96±18.32 vs 1.414±0.424,P<0.05),IL,4 group(9.826 ± 3.152 vs 1.414 ± 0.424,P<0.05)and the anti-IgM group(7.339± 1.633 vs 1.414± 0.424,P<0.05).Fluorescence intensity of BAFF-R was detected by flow cytometry,compared with healthy controls,the surface BAFF-R level of CD19+B cells in ITP patients increased(32.96 ± 9.035 vs 22.73 ± 5.568,P<0.01).The potential downstream target gene of let-7b-5p was found to be ADAM 10 by TargetScan database search.After over-expression of let-7b-5p,ADAM 10 mRNA levels were significantly decreased(0.409±0.123 vs 0.901±0.083,P<0.01).Conversely,inhibition of let-7b-5p expression significantly increased ADAM10 mRNA levels(6.814 ± 2.357 vs 0.901 ± 0.083,P<0.01).A significant negative correlation was found between let-7b-5p level and ADAM10 mRNA level in ITP patient(r=-0.635,P<0.05).The CD61 knockout mice were successfully immunized,and the titer of the anti-CD61 antibody was 1:800.The intraperitoneal injection of 5×104 splenocytes of the immunized mice caused the thrombocytopenia of SCID mice to appear two weeks later.Conclusions:The relative expression levels of miR-3162-3p,let-7b-5p and miR-142-3p in B cells of ITP patients were increased.Among them,let-7b-5p may negatively regulate BAFF-R expression by targeting ADAM10,and may be involved in the survival of autoimmune B cells in ITP patient.Successfully constructed murine ITP model through immuneized CD61 KO mice in our laboratory.