| Objective: To observe the expression levels of miR-146 and its downstream target genes TRAF6,IRAK1 and HIPK3 in the cardiac tissue of mouses by establishment a mouse model of type 1 diabetes cardiomyopathy.The purpose of this study is to analyze the relationship between miR-146 and the development of diabetic cardiomyopathy.For further study on the pathogenesis and clinical treatment of diabetic cardiomyopathy provides a new direction.Methods: 1,Model establishment and grouping: 60 8-week-old C57BL/6J male mice were randomly divided into normal control group(Ctrol,n=30)and the diabetic experimental group(DCM,n=30).The mice in the experimental group were given a five-day continuous intravitoneal injection of 50mg/kg of streptozotocin to induce the construction of diabetic cardiomyopathy animal models,and the mice were fed with a high-fat and high-sugar diet.The normal control group was intraperitoneal injected of equivalent citrate buffer,and the mouse was fed by normal diet.After that,the fasting blood glucose and weight in the experimental group and the control group were monitored regularly every week.At the end of the 12 th weekend,anesthetized mice and took out the heart,weighed the heart weight.2,The morphological changes were observed by HE staining,and the development degree of cardiac fibrosis was detected by Masson staining.3,MRNA expression of miR-146 a,miR-146 b,β-MHC,ANP,IRAK1,TRAF6 and HIPK3 in myocardial tissue were detected by Real-time RT-PCR.4,To detect the protein expression levels of IRAK1 and TRAF6 in the heart by Western blot.5,The targeted relationship between miR-146 and HIPK3 gene: The 3’UTR region sequences of HIPK3 genes and its mutants were inserted respectively into the luciferase report plasmid psiCHECK-2 to construct a wildtype and a mutant recombinant dual luciferase report plasmid.The 293 T cells were divided into 6 groups and transfected with 1)HIPK3-WT +NC negative control;2)HIPK3-WT + mi R-146 a mimics;3)HIPK3-WT + miR-146 b mimics;4)HIPK3-MU + NC negative control;5)HIPK3-MU + miR-146 a mimics;6)HIPK3-MU + miR-146 b mimics respectively,after 48 hours,the luciferase activity in 6 groups of cells was detected.6,statistical analysis: the experiment results used SPSS13.0 statistical software to analysis,and data with arithmetic mean plus or minus standard deviation((x|-) ± s),between groups comparing with independent samples t test,P<0.05 representation of date was statistically significant.Results: 1,Indicators of mouse modeling:(1)blood glucose: the blood glucose in the DCM group was significantly higher than that in Ctrol group(P<0.01).(2)body weight: compared with Ctrol group,the weight of DCM group was significantly decreased(P<0.01).(3)the heart weight: there was no significant change in heart weight in the DCM group compared to the Ctrol group(P>0.05).2,The results of HE staining showed that,compared with the Ctrol group,some cardiac cells in the DCM showed hypertrophy and disordered structure,fibroblast growth is evident.Results of masson staining: the myocardial fibers presented as dark red and the myocardial interstitial collagen presented as dark blue,interstitial collagen fibers were less in the Ctrol group and were mainly distributed around the great vessel wall;there were more collagen fibers around the vascular wall in the heart muscle of the DCM group.3,Real-time RT-PCR detection of mRNA expression: the results showed that miR-146a(P<0.0001)and miR-146b(P<0.0001)in the DCM group decreased significantly compared with the control group.The expression of ANP and β-MHC in DCM group was significantly increased.The mRNA levels of IRAK1(P<0.01)in DCM group was significantly higher than that in Ctrol group.The mRNA level of TRAF6(P<0.05)in DCM group was lower than that in Ctrol group.HIPK3 expression increased in DCM group(P<0.05).4,Western blot results showed that: the expression of IRAK1 protein in the experimental group increased,while the protein expression of TRAF6 decreased.5,The results of dual luciferase report showed that: it was no significant change in luciferase activity after the transfection of 293 T cells with HIPK3-WT and mi R-146 a mimics.It was no significant change in luciferase activity of other three groups of cells transfected with HIPK3-MU.However,after the co-transfection of 293 T cells with HIPK3-WT and miR-146 b mimics,the observation results showed that the luciferase activity decreased(P<0.05).The differences between groups were statistically significant.Conclusions: 1,The data indicated that mi R-146 may participate in the development of diabetic myocardial injury by targeting the IRAK1 mediated inflammatory response.2,There is no targeted relationship between miR-146 a and HIPK3 gene,and miR-146 b may regulate the 3’UTR of HIPK3 gene. |
