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Epigenetic regulation of miR-29a/miR-30c/DNMT3A axis controls SOD2 and mitochondrial oxidative stress in human mesenchymal stem cells.

ABSTRACT: The use of human mesenchymal stem cells (hMSCs) in clinical applications requires large-scale cell expansion prior to administration. However, the prolonged culture of hMSCs results in cellular senescence, impairing their proliferation and therapeutic potentials. To understand the role of microRNAs (miRNAs) in regulating cellular senescence in hMSCs, we globally depleted miRNAs by silencing the DiGeorge syndrome critical region 8 (DGCR8) gene, an essential component of miRNA biogenesis. DGCR8 knockdown hMSCs exhibited severe proliferation defects and senescence-associated alterations, including increased levels of reactive oxygen species (ROS). Transcriptomic analysis revealed that the antioxidant gene superoxide dismutase 2 (SOD2) was significantly downregulated in DGCR8 knockdown hMSCs. Moreover, we found that DGCR8 silencing in hMSCs resulted in hypermethylation in CpG islands upstream of SOD2. 5-aza-2'-deoxycytidine treatment restored SOD2 expression and ROS levels. We also found that these effects were dependent on the epigenetic regulator DNA methyltransferase 3 alpha (DNMT3A). Using computational and experimental approaches, we demonstrated that DNMT3A expression was regulated by miR-29a-3p and miR-30c-5p. Overexpression of miR-29a-3p and/or miR-30c-5p reduced ROS levels in DGCR8 knockdown hMSCs and rescued proliferation defects, mitochondrial dysfunction, and premature senescence. Our findings provide novel insights into hMSCs senescence regulation by the miR-29a-3p/miR-30c-5p/DNMT3A/SOD2 axis.

SUBMITTER: Jung YD

PROVIDER: S-EPMC7509080 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Epigenetic regulation of miR-29a/miR-30c/DNMT3A axis controls SOD2 and mitochondrial oxidative stress in human mesenchymal stem cells.

Jung Yi-Deun YD Park Seul-Ki SK Kang Dayeon D Hwang Supyong S Kang Myoung-Hee MH Hong Seung-Woo SW Moon Jai-Hee JH Shin Jae-Sik JS Jin Dong-Hoon DH You Dalsan D Lee Joo-Yong JY Park Yun-Yong YY Hwang Jung Jin JJ Kim Choung Soo CS Suh Nayoung N

Redox biology 20200909

The use of human mesenchymal stem cells (hMSCs) in clinical applications requires large-scale cell expansion prior to administration. However, the prolonged culture of hMSCs results in cellular senescence, impairing their proliferation and therapeutic potentials. To understand the role of microRNAs (miRNAs) in regulating cellular senescence in hMSCs, we globally depleted miRNAs by silencing the DiGeorge syndrome critical region 8 (DGCR8) gene, an essential component of miRNA biogenesis. DGCR8 kn ...[more]

PMID: 32961441

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Project description:Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by an excessive production of pro-inflammatory cytokines resulting in chronic inflammation and genomic instability. Besides the driver mutations in JAK2, MPL, and CALR genes, the deregulation of miRNA expression may also contribute to the pathogenesis of PMF. To this end, we recently reported the upregulation of miR-382-5p in PMF CD34+ cells. In order to unveil the mechanistic details of the role of miR-382-5p in pathogenesis of PMF, we performed gene expression profiling of CD34+ cells overexpressing miR-382-5p. Among the downregulated genes, we identified superoxide dismutase 2 (SOD2), which is a predicted target of miR-382-5p. Subsequently, we confirmed miR-382-5p/SOD2 interaction by luciferase assay and we showed that miR-382-5p overexpression in CD34+ cells causes the decrease in SOD2 activity leading to reactive oxygen species (ROS) accumulation and oxidative DNA damage. In addition, our data indicate that inhibition of miR-382-5p in PMF CD34+ cells restores SOD2 function, induces ROS disposal, and reduces DNA oxidation. Since the pro-inflammatory cytokine transforming growth factor-β1 (TGF-β1) is a key player in PMF pathogenesis, we further investigated the effect of TGF-β1 on ROS and miR-382-5p levels. Our data showed that TGF-β1 treatment enhances miR-382-5p expression and reduces SOD2 activity leading to ROS accumulation. Finally, inhibition of TGF-β1 signaling in PMF CD34+ cells by galunisertib significantly reduced miR-382-5p expression and ROS accumulation and restored SOD2 activity. As a whole, this study reports that TGF-β1/miR-382-5p/SOD2 axis deregulation in PMF cells is linked to ROS overproduction that may contribute to enhanced oxidative stress and inflammation. Our results suggest that galunisertib may represent an effective drug reducing abnormal oxidative stress induced by TGF-β1 in PMF patients. DATABASE LINKING: GEO: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE103464.

Project description:ObjectiveTo investigate the modulation of microRNAs (miRNAs) upon the neuronal differentiation of mesenchymal stem cells (MSCs) through targeting RE-1 Silencing Factor (REST), a mature neuronal gene suppressor in neuronal and un-neuronal cells.MethodsRat bone marrow derived-MSCs were induced into neuron-like cells (MSC-NCs) by DMSO and BHA in vitro. The expression of neuron specific enolase (NSE), microtubule-associated protein tau (Tau), REST and its target genes, including synaptosomal-associated protein 25 (SNAP25) and L1 cell adhesion molecular (L1CAM), were detected in MSCs and MSC-NCs. miRNA array analysis was conducted to screen for the upregulated miRNAs after neuronal differentiation. TargetScan was used to predict the relationship between these miRNAs and REST gene, and dual luciferase reporter assay was applied to validate it. Gain and loss of function experiments were used to study the role of miR-29a upon neuronal differentiation of MSCs. The knockdown of REST was conducted to show that miR-29a affected this process through targeting REST.ResultsMSCs were induced into neuron-like cells which presented neuronal cell shape and expressed NSE and Tau. The expression of REST declined and the expression of SNAP25 and L1CAM increased upon the neuronal differentiation of MSCs. Among 14 upregulated miRNAs, miR-29a was validated to target REST gene. During the neuronal differentiation of MSCs, miR-29a inhibition blocked the downregulation of REST, as well as the upregulation of SNAP25, L1CAM, NSE and Tau. REST knockdown rescued the effect of miR-29a inhibition on the expression of NSE and Tau. Meanwhile, miR-29a knockin significantly decreased the expression of REST and increased the expression of SNAP25 and L1CMA in MSCs, but did not significantly affect the expression of NSE and Tau.ConclusionmiR-29a regulates neurogenic markers through targeting REST in mesenchymal stem cells, which provides advances in neuronal differentiation research and stem cell therapy for neurodegenerative diseases.

Dataset Information

Epigenetic regulation of miR-29a/miR-30c/DNMT3A axis controls SOD2 and mitochondrial oxidative stress in human mesenchymal stem cells.

Publications

Epigenetic regulation of miR-29a/miR-30c/DNMT3A axis controls SOD2 and mitochondrial oxidative stress in human mesenchymal stem cells.

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