Study Of MiR-669b-3p Regulating IDO Inhibiting Proliferation Of CD4~+T Cells In Vitro

OBJECTIVE:To screen for differentially expressed microRNAs in heterotopic heart transplantation models of mice,investigate the mechanism of the inhibition of CD4+T cell activation by indoleamine 2,3-dioxygenase(IDO)in vitro,and also explore the mechanism of microRNAs affecting immune rejection.METHODS:(1)Mouse heterotopic heart transplantation model was established by microsurgical technique.The donor hearts were screened by microarray.The differentially expressed microRNAs were analyzed and predicted by bioinformatics to find microRNAs targeting IDO gene.The microarray screening results were verified by qRT-PCR.(2)Lentiviruses carrying the microRNA gene were transfected into 3T3 cells and separated into three groups,which are the microRNA over-expression group,the microRNA silencing group and the empty plasmid control group.The expression of microRNA in each group was detected by qRT-PCR,the expression of IDO mRNA in each group was detected by qRT-PCR,and the expression of IDO protein in each group was detected by Western blot.(3)Splenic DC of BALB/c mice was induced and cultured in vitro.Lentivirus carrying IDO gene was transfected into DC,then Western blot was used to detect the expression of IDO protein in DC.(4)Immunomagnetic beads were used to isolate CD4+T cells from C57BL/6 mice,then carried out one-way mixed lymphocyte culture with post-transfection DC.Cell proliferation was detected by CCK-8 method and apoptosis was detected by Annexin-V/PI double staining.RESULTS:(1)The results of microarray screening showed that the expression of 171microRNAs were significantly different,93 of which were significantly up-regulated and 78 were significantly down-regulated.Among them,miR-669b-3p was up-regulated nearly five times and the bioinformatics analysis predicted that IDO might be the target gene.qRT-PCR confirmed that miR-669b-3p was up-regulated by4.7±0.9 times,which was basically consistent with the results of microarray(P<0.05).(2)The expression level of miR-669b-3p in the miR-669b-3p over-expression group detected by qRT-PCR was 10.1±4.8 times higher than in the empty plasmid control group and the expression level of miR-669b-3p in the miR-669b-3p silencing group was 0.3±0.1 times the level in the empty plasmid control group.The expression level of IDO in the over-expression group detected by qRT-PCR was 0.6±0.1 times the level in the empty plasmid control group and in the miR-669b silencing group.The expression level of IDO mRNA was 3.3±1.0 times the level of the control group.Wester blot analysis showed that the ratio of IDO to internal reference gray value was 0.439±0.012 in the miR-669b-3p over-expression group,0.767±0.027 in the miR-669b-3p silencing group,and 0.583±0.016 in the empty plasmid control group.There were significant differences among the groups(P<0.05).(3)Splenic DC of BALB/c mice was induced by differentiation in vitro.Flow cytometry was used to detect the surface antigens CD11c,CD80,CD86 and MHC II of BALB/c mice,which were 89.5%±1.9%,97.0%±2.2%,92.2%±2.8%and87.1%±2.5%,respectively,and accorded with the characteristics of DC surface antigen.After transfection,western blot analysis showed that the ratio of IDO to internal reference gray value was 0.956±0.035 in the IDO~+DC group and 0.725±0.031in empty plasmid DC group.There was significant difference between the two groups(P<0.05).(4)The positive rate of CD4~+T lymphocyte in spleen of C57BL/6 mice was96.1±2.9%by flow cytometry.The results of CCK-8 test in one-way lymphocyte mixed culture showed that the stimulation index of IDO+DC group was 9.5±0.5%,which was lower than that of DC group(11.5±0.6%).The difference was statistically significant(P<0.05).The Annexin-V/PI test showed that the apoptotic rate of IDO~+DC group was 20.9±3.4%,which was higher than that of DC group(10.7±1.3%)and the difference was statistically significant(P<0.05).CONCLUSION:(1)There are significant differences in the expression of multiple microRNAs in the immune rejection of heterotopic heart transplantation of mice.Bioinformatics analysis of miR-669b-3p predicts that IDO may be the target protein.(2)MiR-669b-3p can inhibit the expression of intracellular IDO in vitro.(3)IDO can inhibit the proliferation and increase apoptosis of CD4~+T cells in vitro.(4)MiR-669b-3p may affect CD4~+T cell activity by regulating IDO.