Transforming growth factor-beta (TGF-β), a key mediator of cardiac fibroblast activation, has a major influence on collagen type I production. However, the epigenetic mechanisms by which TGF-β induces collagen type I alpha 1 (COL1A1) expression are not fully understood. This study was designed to examine whether or not DNA methylation is involved in TGF-β-induced COL1A1 expression in cardiac fibroblasts. Cells isolated from neonatal Sprague-Dawley rats were cultured and stimulated with TGF-β1. The mRNA levels of COL1A1 and DNA methyltransferases (DNMTs) were determined via quantitative polymerase chain reaction and the protein levels of collagen type I were determined via Western blot as well as enzyme-linked immunosorbent assay. The quantitative methylation of the COL1A1 promoter region was analyzed using the MassARRAY platform of Sequenom. Results showed that TGF-β1 upregulated the mRNA expression of COL1A1 and induced the synthesis of cell-associated and secreted collagen type I in cardiac fibroblasts. DNMT1 and DNMT3a expressions were significantly downregulated and the global DNMT activity was inhibited when treated with 10 ng/mL of TGF-β1 for 48 h. TGF-β1 treatment resulted in a significant reduction of the DNA methylation percentage across multiple CpG sites in the rat COL1A1 promoter. Thus, TGF-β1 can induce collagen type I expression through the inhibition of DNMT1 and DNMT3a expressions as well as global DNMT activity, thereby resulting in DNA demethylation of the COL1A1 promoter. These findings suggested that the DNMT-mediated DNA methylation is an important mechanism in regulating the TGF-β1-induced COL1A1 gene expression.

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Research has demonstrated that hypoxic preconditioning (HP) can enhance the survival and proliferation of cardiac progenitor cells (CPCs); however, the underlying mechanisms are not fully understood. Here, we report that HP of c-kit (+) CPCs inhibits p53 via the PI3K/Akt-DNMT1 pathway. First, CPCs were isolated from the hearts of C57BL/6 mice and further purified by magnetic-activated cell sorting. Next, these cells were cultured under either normoxia (H0) or HP for 6 hours (H6) followed by oxygen–serum deprivation for 24 hours (24h). Flow cytometric analysis and MTT assays revealed that hypoxia-preconditioned CPCs exhibited an increased survival rate. Western blot and quantitative real-time PCR assays showed that p53 was obviously inhibited, while DNMT1 and DNMT3β were both significantly up-regulated by HP. Bisulphite sequencing analysis indicated that DNMT1 and DNMT3β did not cause p53 promoter hypermethylation. A reporter gene assay and chromatin immunoprecipitation analysis further demonstrated that DNMT1 bound to the promoter locus of p53 in hypoxia-preconditioned CPCs. Together, these observations suggest that HP of CPCs could lead to p53 inhibition by up-regulating DNMT1 and DNMT3β, which does not result in p53 promoter hypermethylation, and that DNMT1 might directly repress p53, at least in part, by binding to the p53 promoter locus.

Atherosclerosis (AS) is a chronic inflammatory disease threatening human health, and vascular smooth muscle cells (VSMCs) are involved in AS processes. Baicalin is a flavonoid compound, which has antiatherosclerotic effect. The aim of our study was to explore the molecular mechanism of baicalin on AS. The expression of miR-126-5p was measured in peripheral blood of AS patients and healthy control. We found miR-126-5p expression was decreased in AS. Then, high-mobility group box 1 (HMGB1) was verified as a target of miR-126-5p and its expression was increased in AS. Similarly, miR-126-5p and HMGB1 expression was downregulated and upregulated in oxidized low-density lipoprotein treated VSMCs (ox-LDL-VSMCs), respectively. Furthermore, baicalin upregulated miR-126-5p and downregulated HMGB1 expression. Functionally, baicalin significantly inhibited ox-LDL-VSMCs proliferation and migration, and miR-126-5p targets HMGB1 to enhance the inhibition induced by baicalin. Taken together, baicalin is able to prevent AS, which suppressed the proliferation and migration of ox-LDL-VSMCs through upregulating miR-126-5p by targeting HMGB1. These findings suggested that baicalin is an effective drug to alleviate AS, and miR-126-5p is a novel therapeutic target for AS.

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