Research On The Expression Profiles Of Treg MiRNAs In Mice Cardiac Transplantation Rejection

ObjectiveThe main purpose of this study was to detect the expression profiles of CD4~+CD25~+Treg microRNAs in mice cardiac transplantation rejection reaction, and then research the microRNA specially regulating the immunosuppressive function of Treg. Further we can provide an effective way of inhibiting graft rejection reaction. Methods1. Established the mice model of abdominal heterotopic heart transplantation, and divided the experimental animals into two groups: homologous transplantation group and allogeneic transplantation group, each group containing 15 animals.2. After the transplantation for 5 days, we separated the CD4~+CD25~+Treg cells of receptor spleen through the magnetic bead and extracted the total RNA from Treg cells, and then finished the concentration and hybridization by microRNA microarray and scanned the slides by Agilent Scanner G2505C(Agilent Technologies). Afterwards, we selected the series of significantly different Treg microRNAs of allogeneic transplantation rejection. At last, we compared the results with the microRNA expression profiles of cardiac xenotrasplantation rejection.3. Selected the significantly different microRNA-146 a and microRNA-451 a of the hybridization results and then made a quantitative analysis by the real-time PCR to verify the resuts of microRNA array.4. We used the microRNAorg, TargetScan and PITA software to predict the target genes of the significantly different microRNA-146 a and microRNA-451 a, and found out the relevant genes corresponding with transplantation rejection reaction, and then we analysed the pathway on which the mi RNA regulated Treg in transplantation rejection reaction.Results1. The successful rate of surgery in the two stages(practicing and stable) of establishing the models were 32.5% and 92.5% respectively and the later chances of success were significantly higher than the former.2. The purity of the CD4~+CD25~+Treg cells of spleen through the magnetic bead was 89.3%, which could be used for further experimental study.3. There were 70 miRNAs in the hybridization results significantly different between the allogeneic transplantation group and the homologous transplantation group, including 58 upregulated miRNAs and 12 down regulated miRNAs. The expression of 9 microRNAs among them was consistent with the microRNA expression profiles of cardiac xenotrasplantation rejection.4. The miRNA-146 a and mi RNA-451 a in Treg cells, with significant difference between the two groups, were analyzed through qRT-PCR. The results showed that the expression of miRNA-146 a in the allogeneic transplantation group was significantly higher than that in the homologous transplantation group and the miRNA-451 a was significantly lower. The change range of miRNAs was consistent with the hybridization results.5. The target genes of miRNA-146 a associated with transplantation rejection reaction were TRAF6,IRAK1 and STAT1, and the target genes of miRNA-451 a were LKB1 and AKT1, predicted by microRNAorg, TargetScan and PITA software. Conclusions1. The expression profiles showed many Treg microRNAs expressed significantly different on the allogeneic transplantation rejection, which could play an important role in cell differentiation, maturation and apoptosis of cells.2. The expression of 9 micro RNAs was consistent with that the microRNA expression profiles between the Treg of allogeneic transplantation rejection and the donor heart of cardiac xenotrasplantation rejection.3. MiRNA-146 a significantly expressed higher and miRNA-451 a dramatically expressed lower in Treg of the allogeneic transplantation rejection, which indicated that they were involved in the process of regulating the Treg cells and thus played a role in the transplantation rejection reaction.