Biomarcadores moleculares na cardiomiopatia diabética: uma abordagem in silico, in vivo e in vitro

Abstract

Hyperglycemia caused by Diabetes mellitus (DM) leads to molecular alterations including mRNA, miRNAs and inflammatory cytokines expression, which are related to the appearance of changes and adaptations characteristic of diabetic cardiomyopathy (DCM). The present study aimed to search through in silico analysis, mRNAs and miRNAs differently expressed in DCM and to validate, in vivo and in vitro, the pathway of regulation of rno-miR-214-3p and its possible target mRNA, phospholipase A2 of the group IIA (Pla2g2a) in conditions of hyperglycemia. Thus, Gene Expression Omnibus microarray databases (GSE4745 and GSE44179) and tools such as Ingenuity Pathway Analysis and TargetScan 7.1 were used to search for possible CMD-related miRNARNAm interactions. In silico, it was observed that Pla2g2a was upregulated and its possible regulatory miRNA rno-miR-214-3p decreased in the left ventricle (LV) of diabetic rats. Thus, in vivo and in vitro approaches using LV samples from Wistar rats with streptozotocin-induced diabetes (40 mg / kg, iv) and in H9c2 cell culture under normoglycemic (NG, 5.5 mmol / L glucose) and hyperglycemic (25 mmol / L of glucose) conditions, and also, transfected with rno-miR-214-3p mimetic in hyperglycemic medium to validate the in silico data. Total RNA and proteins were extracted from the samples and the expression of rno-miR-214-3p, Pla2g2a and Il6 was evaluated by RT-qPCR and Western Blot. In vivo, fibrosis was characterized by increased collagen deposition in the myocardium of diabetic rats (p = 0.023). Hyperglycemia reduced the expression of rnomiR-214-3p (p = 0.043 and p = 0.020) and increased Pla2g2a (p = 0.016 and p = 0.043) and Il6 (p = 0.016 and p = 0.011) gene expression in vivo and in vitro, respectively. Protein expression of sPLA2-IIA (p = 0.011) and IL-6 (p = 0.011) was also increased in LV of diabetic rats. There was a positive correlation between the expression of Pla2g2a, Il6 and hyperglycemia (p < 0.05). Transfection of H9c2 cells with mimetic increased rno-miR214-3p and reduced Pla2g2a and Il6 expression under HG concentrations (50 nM of mimic / 24 h). Therefore, we observed that the expression of rno-miR-214-3p is decreased and its target gene Pla2g2a increased in DM in the three approaches proposed by the present study. In addition, the increase in the expression of rno-miR-214-3p after mimetics transfection caused a decrease in the expression of Pla2g2a and of Il6, indicating a participation of this miRNA in the control of inflammation caused by DM.