H3K14 hyperacetylation‑mediated c‑Myc binding to the miR‑30a‑5p gene promoter under hypoxia postconditioning protects senescent cardiomyocytes from hypoxia/reoxygenation injury

Abstract

Our previous study reported that microRNA (miR)‑30a‑5p upregulation under hypoxia postconditioning (HPostC) exert a protective effect on aged H9C2 cells against hypoxia/reoxygenation injury via DNA methyltransferase 3B‑induced DNA hypomethylation at the miR‑30a‑5p gene promoter. This suggests that miR‑30a‑5p may be a potential preventative and therapeutic target for ischemic heart disease in aged myocardium. The present study aimed to investigate the underlying mechanisms of miR‑30a‑5p transcription in aged myocardium in ischemic heart disease. Cardiomyocytes were treated with 8 mg/ml D‑galactose for 9 days, and then exposed to hypoxic conditions. Cell viability was determined using a cell viability assay. Expression levels of histone deacetylase 2 (HDAC2), LC3B‑II/I, beclin‑1 and p62 were detected via reverse transcription‑quantitative PCR and western blotting. Chromatin immunoprecipitation‑PCR and luciferase reporter assays were performed to evaluate the effect of c‑Myc binding and activity on the miR‑30a‑5p promoter in senescent cardiomyocytes following HPostC. It was found that HPostC enhanced the acetylation levels of H3K14 at the miR‑30a‑5p gene promoter in senescent cardiomyocytes, which attributed to the decreased expression of HDAC2. In addition, c‑Myc could positively regulate miR‑30a‑5p transcription to inhibit senescent cardiomyocyte autophagy. Mechanically, it was observed that increased H3K14 acetylation level exposed to romidepsin facilitated c‑Myc binding to the miR‑30a‑5p gene promoter region, which led to the increased transcription of miR‑30a‑5p. Taken together, these results demonstrated that HDAC2‑mediated H3K14 hyperacetylation promoted c‑Myc binding to the miR‑30a‑5p gene promoter, which contributed to HPostC senescent cardioprotection.

Keywords: autophagy; c‑Myc; hypoxia postconditioning; hypoxia/reoxygenation; histone acetylation; microrna‑30a‑5p.

Figure 1.

HPostC promotes H3K14 hyperacetylation at the miR-30a gene promoter via the downregulation of HDAC2 in senescent cardiomyocytes. (A) The mRNA expression of miR-30a-5p in senescent cardiomyocytes after treatment with TSA. (B and C) The enrichment of H3K14 acetylation at miR-30a-5p gene promoter was identified by ChIP-PCR analysis in the senescent cardiomyocytes. (D and E) The HDAC2 mRNA and protein expression levels in senescent cardiomyocytes. (F and G) HDAC2 binding at the miR-30a-5p gene promoter was analyzed by ChIP-PCR in senescent cardiomyocytes. Data are presented as the mean ± SD from three independent experiments. *P<0.05, **P<0.01. HPostC, hypoxia postconditioning; miR, microRNA; TSA, trichostatin A; HDAC2, histone deacetylase 2; ChIP, chromatin immunoprecipitation; H/R, hypoxia/reoxygenation.

Figure 2.

miR-30a-5p negatively regulated by HDAC2 is involved in HPostC-induced autophagy inhibition. (A and B) The enrichment of H3K14 acetylation at the miR-30a-5p gene promoter was identified by chromatin immunoprecipitation-PCR analysis in cells infected with Ad-shNC and Ad-shHDAC2 for 48 h. (C) miR-30a-5p mRNA expression in senescent cardiomyocytes treated as aforementioned. (D) The relative protein expression levels of LC3B-II/I, BECN1 and p62 after knockdown of HDAC2. (E) The cell viability staining of romidepsin-treated aged H9C2 cell under normoxia, H/R or HPostC conditions (scale bar, 50 µm). Data are presented as the mean ± SD from three independent experiments. *P<0.05, **P<0.01. HPostC, hypoxia postconditioning; miR, microRNA; HDAC2, histone deacetylase 2; H/R, hypoxia/reoxygenation; Ad-, adenovirus; sh, short hairpin RNA; NC, negative control; LC3B, light chain 3β; BECN1, beclin-1.

Figure 3.

c-Myc positively regulates miR-30a-5p transcriptional activity. (A) Schematic diagram shows the locations of the predicted c-Myc binding sites (black hollow circle) surrounded by 5 CpG sites (red vertical bars) in the miR-30a-5p gene core promoter sites (−216/-761). (B) Sequential deletion and substitution mutation analyses identified c-Myc-responsive regions at the miR-30a-5p gene promoter region, miR-30a-5p transcriptional activities in 293T cells were detected using a luciferase reporter assay after solely or serially truncated c-Myc binding sites at the miR-30a-5p promoter region. (C and D) Reverse transcription-quantitative PCR and western blotting were performed to determine c-Myc expression in the senescent cardiomyocytes. Data are presented as the mean ± SD from three independent experiments. *P<0.05, **P<0.01. HPostC, hypoxia postconditioning; miR, microRNA; H/R, hypoxia/reoxygenation.

Figure 4.

Acetylation of H3K14 promotes c-Myc binding to the miR-30a-5p gene promoter to inhibit autophagy of senescent cardiomyocytes. (A) miR-30a-5p mRNA expression was detected after knockdown of c-Myc expression under HPostC treatment. (B) Relative protein detection in the senescent cardiomyocytes treated as aforementioned. (C) Live/dead cell imaging in senescent cardiomyocytes treated as aforementioned (scale bar, 50 µm). (D) Chromatin immunoprecipitation-PCR assay of c-Myc binding at the miR-30a-5p gene promoter. (E) miR-30a-5p mRNA expression was examined by reverse transcription-quantitative PCR. Data are presented as the mean ± SD from three independent experiments. *P<0.05, **P<0.01. HPostC, hypoxia postconditioning; miR, microRNA; H/R, hypoxia/reoxygenation; Ad-, adenovirus; sh, short hairpin RNA; NC, negative control; LC3B, light chain 3β; BECN1, beclin-1.