The Inhibition Of MiR-27b On The Expression Of MCP-1in H9C2Rat Cardiomyocytes

Background:Viral myocarditis (VMC) is a common and serious disease.Our previous study foundthat IL-17can upregulate the expression of mouse cardiomyocytes monocytechemoattractant protein-1(MCP-1).MCP-1-mediated the migration of mononuclear cellsin mice VMC myocardial tissue is an important mechanisms of inflammatory cellinfiltration.microRNA could participate in the regulation of gene transcription bycombined the3’-UTR specific sites of target mRNAs.In this way,microRNA could limitthe translation of the target mRNAs or induce it degradation.In this study,we predictedthat MCP-1may be a target gene of miR-27b by bioinformatics databases,and weinvestigate the effect of miR-27b on the expression of MCP-1induced by IL-17in H9C2rat cardiomyocytes to explore whether miR-27b downregulate the expression of MCP-1through further research.Methods:1. The experimental design: The H9C2myocardial cell lines were subcultured. Thegroups were divided in the three ways.1) There were six time-response groups including0h,2h,4h,8h,12h,24h after IL-17treatment in the study of effect of IL-17on the expression of MCP-1.2) To analyze transfection efficiency of miR-27b mimics in H9C2cardiomyocytes,there were three groups including miR-27b mimics transfection group, miRNA mimics negative transfection group and blank control group.3) When the role of miR-27b in the expression of MCP-1was investigated, therewere four groups including miR-27b mimics intervention group treated with miR-27bmimics and IL-17, miRNA negative control group treated with miRNA mimics negativecontrol and IL-17, IL-17group and the blank control group.2. miR-27b mimics was transfected into the H9C2cardiomyocytes bysiRNA-MateTM.3. The expression of miR-27b and MCP-1mRNA in H9C2cardiomyocytes wasdetected by SYBR green I fluorescence quantitative RT-PCR.4. Enzyme-linked immunosorbent assay (ELISA) was used to analyze the proteinconcentration of MCP-1in the culture supernatant of the H9C2cardiomyocytes.5. SPSS17.0statistical software was applied for the data statistics.Multiple sets ofmeasurement data were compared with single factor analysis of variance and the twogroups were compared with LSD-t test. A value of P <0.05was considered to bestatistically significant.Results:1. The effect of IL-17on the expression of MCP-1in the H9C2cells: MCP-1mRNA expression began to increase at2h after stimulated by IL-17, reached a peak at4h,later began to decline, and the amount of MCP-1protein expression began to increase at2h,after that it increased gradually in2~24h.2. The analysis of miR-27b after transfected with miR-27b mimics in H9C2cardiomyocytes:compared with miRNA mimics negative control group, the quantity ofmiR-27b was significantly increased in the group which transfection of miR-27b mimics.Compared with the blank group, the level of miR-27b was no significant change in thegroup which transfected the miRNA mimics negative control.3. The effect of miR-27b on the expression of MCP-1induced by IL-17: MCP-1mRNA and protein levels were significantly increased in the H9C2cells stimulated by IL-17as compared with the blank group(P <0.05); while there were no differences in theexpression of MCP-1mRNA and protein in IL-17group as compared with miRNAmimics negative control group (P0.05); the miR-27b mimics intervention groupcompared with that of miRNA mimics negative control group, MCP-1mRNA andprotein levels were significantly decreased (P<0.01).Conclusion:1. IL-17could up-regulate the expression of MCP-1mRNA and protein in H9C2cardiacmyocytes.2. IL-17could induce the expression of MCP-1protein in the cardiacmyocytes intime-dependent manners.3. miR-27b might down-regulate the expression of MCP-1which induced by IL-17in H9C2cardiacmyocytes.