Ischemic Postconditioning Protects against Aged Myocardial Ischemia/Reperfusion Injury by Transcriptional and Epigenetic Regulation of miR-181a-2-3p

Figure 3

IPostC inhibits autophagy by DNA hypomethylation of miR-181a-2-3p in aged myocardium. (a) The promoter activity of miR-181a-2-3p was evaluated by the luciferase reporter assay. Different deletion fragments of miR-181a-2-3p promoter (-2000/+117, -1200/+117, -600/+117, and -200/+117) cotransfected into HEK293 cells with Renilla luciferase vector (internal control), and the results were represented as firefly luciferase activity normalized to Renilla luciferase activity (). (b) DNA methylation levels of miR-181a-2-3p promoter were detected by nMS-PCR in aged myocardium after IPostC (). The reactions for unmethylated and methylated DNA are denoted by U and M, respectively. (c) MassARRAY analysis was performed to determine the methylation levels of miR-181a-2-3p promoter from -2000 to +117 in aged cardiomyocytes after HPostC. Each circle represents a single CpG analyzed. Methylation frequencies are displayed in a color code that extends from yellow (lower methylation frequencies) to blue (higher methylation frequencies). White circles indicate not analyzed methylation values due to CpGs with high or low mass Dalton peaks falling outside the conservative window of reliable detection for the EpiTYPER software. (d) miR-181a-2-3p expression was validated by qRT-PCR in aged cardiomyocytes treated with DC_05 (DNMT1 inhibitor), Theaflavin-3 (TF-3, DNMT3a inhibitor), and Nanaomycin A (NA, DNMT3b inhibitor) after HPostC, respectively (). (e) DNMT3b expression was detected by Western blot both in aged myocardium after IPostC () and in aged cardiomyocytes after HPostC (). (f) The expression of miR-181a-2-3p was analyzed by qRT-PCR in aged cardiomyocytes transfected with sh-DNMT3b after HPostC (). (g) MassARRAY analysis was used to measure the methylation levels of miR-181a-2-3p promoter in aged cardiomyocytes transfected with sh-DNMT3b after HPostC. (h) Western blot analysis of LC3B-II and p62 expression in aged cardiomyocytes transfected with sh-DNMT3b in the presence of CQ (). Data were presented as the . .

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)