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Epigenetic Modification of MicroRNA-200b Contributes to Diabetic Vasculopathy.

ABSTRACT: Hyperglycemia (HG) induces genome-wide cytosine demethylation. Our previous work recognized miR-200b as a critical angiomiR, which must be transiently downregulated to initiate wound angiogenesis. Under HG, miR-200b downregulation is not responsive to injury. Here, we demonstrate that HG may drive vasculopathy by epigenetic modification of a miR promoter. In human microvascular endothelial cells (HMECs), HG also lowered DNA methyltransferases (DNMT-1 and DNMT-3A) and compromised endothelial function as manifested by diminished endothelial nitric oxide (eNOS), lowered LDL uptake, impaired Matrigel tube formation, lower NO production, and compromised VE-cadherin expression. Bisulfite-sequencing documented HG-induced miR-200b promoter hypomethylation in HMECs and diabetic wound-site endothelial cells. In HMECs, HG compromised endothelial function. Methyl donor S-adenosyl-L-methionine (SAM) corrected miR-200b promoter hypomethylaton and rescued endothelial function. In vivo, wound-site administration of SAM to diabetic mice improved wound perfusion by limiting the pathogenic rise of miR-200b. Quantitative stable isotope labeling by amino acids in cell culture (SILAC) proteomics and ingenuity pathway analysis identified HG-induced proteins and principal clusters in HMECs sensitive to the genetic inhibition of miR-200b. This work presents the first evidence of the miR-200b promoter methylation as a critical determinant of diabetic wound angiogenesis.

SUBMITTER: Singh K

PROVIDER: S-EPMC5768662 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Epigenetic Modification of MicroRNA-200b Contributes to Diabetic Vasculopathy.

Singh Kanhaiya K Pal Durba D Sinha Mithun M Ghatak Subhadip S Gnyawali Surya C SC Gnyawali Surya C SC Khanna Savita S Roy Sashwati S Sen Chandan K CK

Molecular therapy : the journal of the American Society of Gene Therapy 20170912 12

Hyperglycemia (HG) induces genome-wide cytosine demethylation. Our previous work recognized miR-200b as a critical angiomiR, which must be transiently downregulated to initiate wound angiogenesis. Under HG, miR-200b downregulation is not responsive to injury. Here, we demonstrate that HG may drive vasculopathy by epigenetic modification of a miR promoter. In human microvascular endothelial cells (HMECs), HG also lowered DNA methyltransferases (DNMT-1 and DNMT-3A) and compromised endothelial func ...[more]

PMID: 29037594

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Project description:BACKGROUND:Diabetes mellitus is characterized by chronic vascular disorder and presents a main risk factor for cardiovascular mortality. In particular, hyperglycaemia and inflammatory cytokines induce vascular circulating tissue factor (TF) that promotes pro-thrombotic conditions in diabetes. It has recently become evident that alterations of the post-transcriptional regulation of TF via specific microRNA(miR)s, such as miR-126, contribute to the pathogenesis of diabetes and its complications. The endothelial miR-19a is involved in vascular homeostasis and atheroprotection. However, its role in diabetes-related thrombogenicity is unknown. Understanding miR-networks regulating procoagulability in diabetes may help to develop new treatment options preventing vascular complications. METHODS AND RESULTS:Plasma of 44 patients with known diabetes was assessed for the expression of miR-19a, TF protein, TF activity, and markers for vascular inflammation. High miR-19a expression was associated with reduced TF protein, TF-mediated procoagulability, and vascular inflammation based on expression of vascular adhesion molecule-1 and leukocyte count. We found plasma expression of miR-19a to strongly correlate with miR-126. miR-19a reduced the TF expression on mRNA and protein level in human microvascular endothelial cells (HMEC) as well as TF activity in human monocytes (THP-1), while anti-miR-19a increased the TF expression. Interestingly, miR-19a induced VCAM expression in HMEC. However, miR-19a and miR-126 co-transfection reduced total endothelial VCAM expression and exhibited additive inhibition of a luciferase reporter construct containing the F3 3'UTR. CONCLUSIONS:While both miRs have differential functions on endothelial VCAM expression, miR-19a and miR-126 cooperate to exhibit anti-thrombotic properties via regulating vascular TF expression. Modulating the post-transcriptional control of TF in diabetes may provide a future anti-thrombotic and anti-inflammatory therapy.

Project description:The present study aimed to investigate the expression of miR-200b and protein kinase Cα (PKCα) in pituitary tumors and to determine whether miR-200b may inhibit proliferation and invasion of pituitary tumor cells. The regulation of PKCα expression was targeted in order to find novel targets for the treatment of pituitary tumors. In total, 53 pituitary tumor tissue samples were collected; these included 28 cases of invasive pituitary tumors and 25 cases of non-invasive tumors, in addition to 5 normal pituitaries. The expression level of miR-200b in the pituitary tumor tissue was detected by quantitative polymerase chain reaction (qPCR) and the expression of PKCα protein was detected by immunohistochemistry. A PKCα 3'untranslated region (UTR) luciferase vector was constructed and a dual luciferase reporter gene assay was employed in order to examine the effect of miR-200b on the PKCα 3'UTR luciferase activity. AtT-20 cells were transfected with miR-200b mimics, PKCα siRNA and miR-200b mimics + PKCα, and the changes in cellular proliferation, invasion and apoptosis were observed via MTT, Transwell assay and flow cytometric analysis. Furthermore, PKCα mRNA expression was determined by qPCR, and Western blotting was performed to detect the expression of PKCα protein. miR-200b revealed downregulation in invasive pituitary tumor tissue, and the expression level was significantly down-regulated compared with normal and non-invasive pituitary tumor tissue (P<0.01). In addition, the positive rate of PKCα protein expression in invasive pituitary tumor tissues was significantly higher than in normal and non-invasive tissues (P<0.01). PKCα protein levels are inversely correlated with miR-200b levels in invasive pituitary tumor tissues (r=-0.436, P=0.021). The dual luciferase reporter gene assay revealed that miR-200b could specifically bind to the 3'UTR of PKCα and significantly inhibit the luciferase activity by 39% (P<0.01). Upregulation of miR-200b or downregulation of PKCα could suppress cell proliferation and invasion, and increase apoptosis of AtT-20 cells. It was revealed that PKCα siRNA could suppress both proliferation and invasion of AtT-20 cells and partially simulate the function of miR-200b. Expression of PKCα mRNA and protein decreased significantly in AtT-20 cells overexpressing miR-200b. Additionally, miR-200b was significantly down-regulated in invasive pituitary tumor tissue and inversely correlated with PKCα protein levels. In conclusion, miR-200b inhibited proliferation and invasiveness and promoted the apoptosis of pituitary tumor cells by targeting PKCα. The observations of the present study indicate that miR-200b and PKCα may serve as promising therapeutic targets for invasive pituitary tumors.

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Epigenetic Modification of MicroRNA-200b Contributes to Diabetic Vasculopathy.

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Epigenetic Modification of MicroRNA-200b Contributes to Diabetic Vasculopathy.

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