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Microrna-217 modulates human skin fibroblast senescence by directly targeting DNA methyltransferase 1.

ABSTRACT: DNA methyltransferase 1 (DNMT1) is a major epigenetic regulator associated with many biological processes. However, the roles and mechanisms of DNMT1 in skin aging are incompletely understood. Here we explored the role of DNMT1 in human skin fibroblasts senescence and its related regulatory mechanisms. DNMT1 expression decreased in passage-aged fibroblasts and DNMT1 silencing in young fibroblasts induced the senescence phenotype. MiR-217 is predicted to target DNMT1 mRNA and miR-217 expression increased in passage-aged fibroblasts. MiR-217 directly targeted the 3'-untranslated region (3'-UTR) of DNMT1 in HEK 293T cells and inhibited DNMT1 expression in fibroblasts. MiR-217 overexpression induced a senescence phenotype in young fibroblasts, and miR-217 downregulation in old HSFs partially reversed the senescence phenotype. However, these effects could be significantly rescued by regulating DNMT1 expression in fibroblasts. After regulating miR-217 levels, we analyzed changes in the promoter methylation levels of 24 senescent-associated genes, finding that 6 genes were significantly altered, and verified p16 and phosphorylated retinoblastoma (pRb) protein levels. Finally, an inverse correlation between DNMT1 and miR-217 expression was observed in skin tissues and different-aged fibroblasts. Together, these findings revealed that miR-217 promotes fibroblasts senescence by suppressing DNMT1-mediated methylation of p16 and pRb by targeting the DNMT1 3'-UTR.

SUBMITTER: Wang B

PROVIDER: S-EPMC5464883 | biostudies-other | 2017 May

REPOSITORIES: biostudies-other

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Microrna-217 modulates human skin fibroblast senescence by directly targeting DNA methyltransferase 1.

Wang Ben B Du Rui R Xiao Xiao X Deng Zhi-Li ZL Jian Dan D Xie Hong-Fu HF Li Ji J

Oncotarget 20170501 20

DNA methyltransferase 1 (DNMT1) is a major epigenetic regulator associated with many biological processes. However, the roles and mechanisms of DNMT1 in skin aging are incompletely understood. Here we explored the role of DNMT1 in human skin fibroblasts senescence and its related regulatory mechanisms. DNMT1 expression decreased in passage-aged fibroblasts and DNMT1 silencing in young fibroblasts induced the senescence phenotype. MiR-217 is predicted to target DNMT1 mRNA and miR-217 expression i ...[more]

PMID: 28380423

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Project description:BackgroundRecent studies have highlighted the contribution of senescent mesenchymal and epithelial cells in Idiopathic Pulmonary Fibrosis (IPF), but little is known regarding the molecular mechanisms that regulate the accumulation of senescent cells in this disease. Therefore, we addressed the hypothesis that the loss of DNA repair mechanisms mediated by DNA protein kinase catalytic subunit (DNA-PKcs) in IPF, promoted the accumulation of mesenchymal progenitors and progeny, and the expression of senescent markers by these cell types.MethodsSurgical lung biopsy samples and lung fibroblasts were obtained from patients exhibiting slowly, rapidly or unknown progressing IPF and lung samples lacking any evidence of fibrotic disease (i.e. normal; NL). The expression of DNA-Pkcs in lung tissue was assessed by quantitative immunohistochemical analysis. Chronic inhibition of DNA-PKcs kinase activity was mimicked using a highly specific small molecule inhibitor, Nu7441. Proteins involved in DNA repair (stage-specific embryonic antigen (SSEA)-4+ cells) were determined by quantitative Ingenuity Pathway Analysis of transcriptomic datasets (GSE103488). Lastly, the loss of DNA-PKc was modeled in a humanized model of pulmonary fibrosis in NSG SCID mice genetically deficient in PRKDC (the transcript for DNA-PKcs) and treated with Nu7441.ResultsDNA-PKcs expression was significantly reduced in IPF lung tissues. Chronic inhibition of DNA-PKcs by Nu7441 promoted the proliferation of SSEA4+ mesenchymal progenitor cells and a significant increase in the expression of senescence-associated markers in cultured lung fibroblasts. Importantly, mesenchymal progenitor cells and their fibroblast progeny derived from IPF patients showed a loss of transcripts encoding for DNA damage response and DNA repair components. Further, there was a significant reduction in transcripts encoding for PRKDC (the transcript for DNA-PKcs) in SSEA4+ mesenchymal progenitor cells from IPF patients compared with normal lung donors. In SCID mice lacking DNA-PKcs activity receiving IPF lung explant cells, treatment with Nu7441 promoted the expansion of progenitor cells, which was observed as a mass of SSEA4+ CgA+ expressing cells.ConclusionsTogether, our results show that the loss of DNA-PKcs promotes the expansion of SSEA4+ mesenchymal progenitors, and the senescence of their mesenchymal progeny.

Project description:BackgroundThe homologous recombination (HR) pathway is involved in DNA damage response (DDR), which is crucial to cancer cell survival after treatment with DNA damage agents. O6-methylguanine DNA methyltransferase (MGMT) is associated with cisplatin (CDDP) resistance in cancer cells; however, the underlying mechanisms remain unclear. Here, we explored the interactions between MGMT and the HR pathway in CDDP-activated DDR and their clinical implications in nasopharyngeal carcinoma (NPC).MethodsHuman NPC cells were assessed using loss-of-function approaches in vitro. The expression correlations between MGMT and major proteins of the HR pathway were analyzed through Western blotting, quantitative real-time PCR, and bioinformatic analysis by using a public database. The physical interactions between MGMT and HR proteins were studied using co-immunoprecipitation and immunofluorescence analyses. Cell comet tails and γ-H2AX expression levels were examined to evaluate double-strand break (DSB) formation. Established immunofluorescence and reporter analyses were conducted to measure HR activity. Xenograft and cell viability studies were used to assess the therapeutic potential of MGMT inhibition in combination with CDDP and poly(ADP-ribose) polymerase (PARP) inhibitor, respectively.ResultsAmong major proteins of the HR pathway, MGMT suppression inhibited CDDP-induced RAD51 expression. Bioinformatic analyses showed a positive correlation between MGMT and RAD51 expression in patients with NPC. Moreover, MGMT physically interacted with BRCA1 and regulated CDDP-induced BRCA1 phosphorylation (ser 988). In functional assays, MGMT inhibition increased CDDP-induced DSB formation through attenuation of HR activity. NPC xenograft studies demonstrated that MGMT inhibition combined with CDDP treatment reduced tumor size and downregulated RAD51 expression and BRCA1 phosphorylation. Furthermore, MGMT suppression increased PARP inhibitor-induced cell death and DSB formation in NPC cells.ConclusionMGMT is crucial in the activation of the HR pathway and regulates DDR in NPC cells treated with CDDP and PARP inhibitor. Thus, MGMT is a promising therapeutic target for cancer treatments involving HR-associated DDR.

Dataset Information

Microrna-217 modulates human skin fibroblast senescence by directly targeting DNA methyltransferase 1.

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Microrna-217 modulates human skin fibroblast senescence by directly targeting DNA methyltransferase 1.

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