CircRNA Expression Regulating Pyroptosis Of The Heart Tissue Induced By Diabetic Mellitus

Objective: This research mainly was to explore circular RNA in diabetic mice,which related to pyroptosis of myocardial cell and its possible mechanism of diabetic cardiomyopathy.Methods: Each of the 10 mice was used for animal experiments in the control(Wild type,WT)group and the model(db / db)group performed weight detection and fasting glucose monitoring.The primary cells of the mice were stimulated with high glucose.They were divided into the low glucose group(5.5m M glucose),the mannitol group(24.5m M mannitol + 5.5m M glucose)and the high glucose group(30m M glucose).After 48 h,total RNA was extracted.Cultured mouse fibroblasts NIH3T3,these cells were seeded into 6-well plates.The blank control group,negative plasmid group and circRNA overexpression group were cultured.After the cell transfected for 12 hours,it was replaced with a new culture medium,and continued to cultivate for 48 h,total RNA was also extracted.Doppler was used to evaluate the cardiac function,in addition,H&E and WGA staining were introduced for myocardial remodeling.Immunohistochemistry was used to observe the distribution of caspase-1 and IL-1β in the heart.The expression of caspase-1and IL-1β in mice left ventricles were detected by Western blot.TUNEL(terminal deoxynucleotidyl transferase-mediated d UTP-biotin nick end labeling)was used to detect DNA fragmentation.The screening of the differentially expressed circRNAs were performed by circRNA chip analysis in hearts of diabetic mice,the expression of SIRT1 and circRNA was detected by q PCR.Results: Compared WT group mice at same age,their body weight and fasting blood glucose significantly increased in db / db group.The echocardiography showed their ventricular wall thickened in db / db group,but heart cavity became smaller.The mouse ejection fraction and fractional shortening increased to70.63% and 45.47%.The stroke volume decreased,and the contraction and end-diastolic volume decreased,too.The end-diastolic volume decreased mainly.The weight / tibial length of db / db mice were larger than that of the control group.HE staining showed that the myocardial cells in the control group were neatly arranged and the nuclei were uniform and the cytoplasm was evenly stained.The cells in the db / db group compared with the control group were hypertrophied,irregularly arranged,irregular in morphology and uneven in the size of the nucleus.WGA staining also indicated myocyto hypertrophy in db/db mice.The expression of caspase-1 and IL-1β related to pyroptosis protein in theheart tissue of db / db mice increased.There was significant difference.TUNEL was positive.There were 792 circRNAs expressed in the two groups,25 of which were up-regulated by 1.5 times or more,and the circRNA group was up to 1.5 times by chip detection.The higher abundance of mm9＿circ＿000807,mm9＿circ＿010964,mm9＿circ＿008009,mm9＿circ＿011728 were resistant to RNase R.Real-time quantitative PCR detection confirming mm9＿circ＿013874 and mm9＿circ＿008009 were down-regulated,but mm9＿circ＿008009 down-regulation was more pronounced about4-fold,and was statistically significant.Arraystar software predicted that the mi RNAs adsorbed by mm9＿circ＿008009 were mi R-224-5p,mi R-7022-3p,mi R-7683-3p,mi R-471-3p and mi R-6371,respectively.There were three binding sites on average.Biological software predicted that mi R-471-3p binded to the SIRT1 3’UTR end and had two binding sites.SIRT1 was down-regulated in the primary cells of neonatal mice treated with high glucose,and SIRT1 was up-regulated in NIH3T3 cells that were transfected with mm9＿circ＿008009 plasmid.Conclusion: The differentially expression of circRNA was related to the pathogenesis of diabetic cardiomyopathy and mm9＿circ＿008009 presented the most obvious reaction and may regulate pyroptosis through mi R-471-3p/SIRT1 in DCM.