Expression Profile Of MiRNAs In Patients With Immune Thrombocytopenia And Role Of MiR-148a

Part One Screen the expression profiles of mi RNAs involved in immunethrombocytopenia patients' peripheral monocytesIn this part,we will screen out the differentially expressed miRNAs that are associated with the pathogenesis of Immune Trombocytopenia(ITP).The peripheral blood mononuclear cells were isolated by density gradient centrifugation,and the miRNAs and mRNA expression profiles of ITP patients were analyzed by high throughput microarray.The significantly differentially expressed miRNAs related to the pathogenesis of ITP were selected for a further study,and its expression was verified by real-time quantitative PCR.The results showed that there were 216 significant up-regulated miRNAs and 150 down-regulated miRNAs in the chronic group compared with the normal control group.And there were 55 significant up-regulated mi RNAs and 45 down-regulated miRNAs in the chronic group compared with the newly diagnosed group.We selected the significantly differentially expressed miRNAs in the PBMCs of ITP patients from microarray,combining with the immune-related miRNAs reported in other literature articles,miR-148 a was screened out finally.The expression of miR-148 a was detected by real-time quantitative PCR in twenty-one newly diagnosed ITP patients,twenty-four chronic ITP patients and healthy controls respectively,admitted from Shanghai ChangHai hospital and 100 Military hospital.Compared with that in PBMCs of healthy controls,the expression of miR-148 a in newly diagnosed ITP patients and chronic ITP patients was up-regulated.Moreover,the expression of mi R-148 a in newly diagnosed ITP patients and chronic ITP patients was also different.The result is consistent with that of miRNA expression profiles,indicating that the results of microarray are reliable.At the same time,the level of Th1 and Th2 related cytokines in the plasma of ITP patients were detected by enzyme-linked immunosorbent assay(ELISA).This part results indicated that ITP patients may have specific miRNAs expression profiles,and miRNAs expression is different in different stages of ITP patients,indicating that miRNAs is involved in the pathogenesis of ITP.Furthermore,the expression of miR-148 a in different ITP stage is distinct,suggesting that miR-148 a could be used as a potential biomarker.Meanwhile,there is a polarization state of Th1 cells in ITP patients.Part Two Preliminary study on the function of mi R-148 a inimmune thrombocytopeniaIn the first part of the study,we screened and validated that miR-148 a is associated with the pathogenesis of ITP.In this part,in order to further elucidate the specific mechanism of miR-148 a in the pathogenesis of ITP,we prepared to screen out the target gene of miR-148 a first through bioinformatics analysis together with the mRNA array datas from first part.Secondly,we used miR-148 a mimics / inhibitors to transfect PBMCs of healthy people,and verified the expression of miR-148 a by RT-PCR.Finally,miR-148 a mimics / inhibitors was used to transfect PBMCs of ITP patients,followed by the stimiulation of LPS.The expression of Th1 and Th2-related cytokines was verified by RT-PCR.The results showed that the expression of Rel A mRNA was significantly decreased after overexpression of miR-148 a compared with negative control.In contrast,the expression was significantly up-regulated when miR-148 a was inhibited.At the same time,the levels of Th1-related cytokines IL-8,IL-1b and TNF-a were slightly up-regulated in miR-148 a overexpressed PBMCs,after the stimiulation of LPS,compared to miR-148 a negative controls.However,Th2-related cytokines IL-4,IL-5,IL-10 levels were significantly decreased.Moreover,the expression of Th1-associated cytokines IL-8,IL-1b and TNF-a were still slightly up-regulated after LPS stimulation,and Th2-related cytokines IL-4,IL-5,IL-10 levels were significantly increased in mi R-148 a inhibited PBMCs.This part suggests that mi R-148 a may play an important role in the pathogenesis of ITP by paticipating the NF-?B signaling pathway to inhibit the secretion of Th2-related cytokines,and promote the Th1 polarization through inhibiting the expression of RelA.