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DNMT1-repressed NSun2 Expression Wires DNA Cytosine Methylation and mRNA Cytosine Methylation to Modulate Osteosarcoma Apoptosis

ABSTRACT: Background: Cellular apoptosis is a central mechanism the chemotherapy leveraged to treat human cancers. Cytosine methylations installed on both DNA and mRNA are documented to regulate apoptosis independently. However, the interplay or crosstalk between them in cellular apoptosis has not yet been explored. Methods: Cisplatin and doxorubicin were used to induce cell apoptosis in three differed types of osteosarcoma cell lines, respectively. Bisulfite-PCR, MS-PCR, and MeDIP experiments were employed to evaluate promoter cytosine methylation, and MeRIP were performed to investigate mRNA cytosine methylation. MeRIP-seq revealed the common target genes regulated by DNMT1 and NSun2. Particular chimeric reporter constructs were generated and dual luciferase assays dissected corresponding regulations via DNA and/or mRNA methylation. Cell viability assay, apoptosis analysis by flow cytometry, and tumor xenograft model were utilized to determine osteosarcoma drug resistance. Results: Promoter methylation by DNMT1 dialogues with mRNA methylation by NSun2 to coordinately regulate osteosarcoma cell apoptosis. DNMT1 was induced in osteosarcoma cell apoptosis triggered by chemotherapeutic drugs, whereas NSun2 expression was dampened. DNMT1 was found to represses NSun2 expression by methylating NSun2 promoter. Moreover, DNMT1 and NSun2 regulate anti-apoptotic genes AXL, NOTCH2, and YAP1 through DNA and mRNA methylation, respectively. Upon exposure to cisplatin or doxorubicin, DNMT1 elevation drastically reduced these anti-apoptotic gene expressions via enhanced promoter methylation coupled with NSun2 ablation-mediated attenuation of mRNA methylation, thus rendering osteosarcoma to apoptosis. Conclusions: Our findings establish a crosstalk of importance between DNA and RNA cytosine methylations in determining osteosarcoma resistance to apoptosis during chemotherapy, shedding new light on future treatment of osteosarcoma, and add additional layers to the control of gene expression at different epigenetic levels.

ORGANISM(S): Homo sapiens

PROVIDER: GSE205312 | GEO | 2025/06/01

REPOSITORIES: GEO

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Project description:Aims: Mammalian mRNA internal modification m6A has been suggested to be essential in the maintenance of cardiac homeostasis. The role of mRNA cytosine methylation (m5C) in the heart remains unexplored.Methods and results: In contrast with human normal heart, the expression of RNA cytosine methyltransferase NSun2 was found to be significantly elevated in the hypertrophied one. Consistently, NSun2 expression was increased in hypertrophic rat ventricular myocytes induced in vitro, as well as in TAC (transverse aortic constriction) developed hypertrophied murine heart. Cardiomyocyte-specific knockout of NSun2 (Myh6-CreERT2, NSun2 flox+/+) dramatically accelerated heart failure progression in TAC mice. RNA m5C immunoprecipitation followed by sequencing and MeRIP (methylation-specific RNA immunoprecipitation) revealed that NSun2 particularly methylates the mRNA of protein kinase A (PKA) catalytic subunit alpha (PRKACA). Polyribosome and dual luciferase reporter gene analysis further suggested that NSun2 promotes PRKACA translation via RNA methylation. In TAC hypertrophic cardiomyocytes, NSun2 ablation markedly dampened PKA signaling, as evidenced by reduced PKA activity and protein phosphorylation levels of PKA diverse substrates, impaired myocyte contraction and relaxation, and disturbed calcium transients. Cardiac-restricted overexpression of NSun2 via adeno-associated virus 9 (AAV9, NSun2 expression cassette driven by cTnT promoter) sensitized heart hypertrophic response, whereas administration of PKA inhibitor H89 successfully blocked this process, reiterating the importance of NSun2-PRKACA regulation in cardiac hypertrophic program.Conclusions: Our study showed that RNA methyltransferase NSun2 promotes PRKACA translation via RNA methylation, whereby achieving its modulation of PKA signaling within the cardiomyocyte. NSun2-PRKACA regulation is required for cardiac homeostasis maintenance and adaptation to hypertrophic stresses. These findings furnish us with novel understanding of heart function mediated by mRNA cytosine methylation in cardiac homeostasis.

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DNMT1-repressed NSun2 Expression Wires DNA Cytosine Methylation and mRNA Cytosine Methylation to Modulate Osteosarcoma Apoptosis

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