MiRNA-21 Mediates The Antiangiogenic Activity Of Metformin Through Targeting PTEN And SMAD7 Expression And PI3K/AKT Pathway

Objective: Metformin is an anti-diabetic drug commonly used for type? diabetes therapy.Recent studies demonstrated that metformin exhibits antiangiogenic activity through inhibiting the proliferation and migration of endothelial cells(ECs)beyond the protective effects of lower blood pressure and cardiovascular.However,the precise regulatory mechanisms are still unclear.Micro R-21(miR-21)is widely expressed in a variety of tissues,and involved in the development of kinds of cardiovascular diseases.Mi R-21 plays a key role in angiogenesis by targeting multiple genes in regulation of the proliferation,migration and apoptosis of ECs.We tested the hypothesis that metformin is involved in the regulation of anti-angiogenesis by down-regulating the expression of miR-21.Methods: 1.Using q RT-PCR,we examined the expression levels of miR-21 in human umbilical vein endothelial cells(HUVECs)treated with metformin or vehicle control in a dose and time manner.2.HUVECs were treated with metformin after transfected with miR-21 mimics,inhibitors and negative control(NC),respectively.The proliferation was measured by CCK-8 assay,and migration was evaluated by wound healing test.Furthermore,formation of tubules by HUVECs was studied by using 24-well plates coated with growth-factor-reduced Matrigel.3.Bioinformatic analysis and identification of miRNA targets were assessed by using luciferase assays.4.The expression levels of PTEN and SMAD7 m RNA and protein were detected by q RT-PCR and western blot respectively after the HUVECs were transfected with miR-21 mimics,inhibitors and negative control(NC).5.We measured the expression of PTEN,TGF-?1 and SMAD7 as well as the phosphorylation level of Akt,ERK1/2 and SMAD2/3 after the HUVECs were treated with metformin and miR-21 mimics,inhibitors.We used a potent small-molecule antagonist(LY2157299)of TGF-? receptor I kinase(TGF-? R1)to evaluate the role of TGF-? in the regulation of miR-21 and its target,and further examine the regulatory mechanism of miR-21 in metformin-induced angiogenic activity.Results: 1.The level of miR-21 in HUVECs was significantly downregulated by metformin in a dose-and time-dependent manner with a peak at 20 m M and 24 h.2.Metformin treatment could significantly inhibit the proliferation and migratory of HUVECs.Furthermore,in presence of metformin,the down-regulation of miR-21 by inhibitor caused a significantly stronger inhibition of HUVECs proliferation and migratory.Conversely,the overexpression of miR-21 by miR-21 mimic significantly abrogated metformin-mediated inhibition of HUVECs proliferation and migratory.3.Bioinformatic analysis and luciferase assays have shown that PTEN and SMAD7 were the direct target gene of miR-21.4.Both PTEN and SMAD7 m RNA/protein levels were significantly increased by miR-21.5.The upregulation of PTEN and SMAD7 m RNA/protein levels by metformin is through the downregulation of TGF-?-induced miR-21.6.Metformin treatment could significantly inhibit the phosphorylations of Akt,ERK1/2 and SMAD2/3.Furthermore,the down-regulation of miR-21 by inhibitor caused a significantly stronger inhibition of phosphorylations of Akt,ERK1/2 and SMAD2/3 in presence of metiform.Conversely,the overexpression of miR-21 by miR-21 mimic significantly abrogated metformin-mediated inhibition of phosphorylations of Akt,ERK1/2 and SMAD2/3.7.Tubule formation assays revealed that metformin treatment could significantly inhibit the ability of HUVECs tubule formation.The down-regulation of miR-21 by inhibitor caused a significantly stronger inhibition of the ability of HUVECs tubule formation in presence of metiform.Conversely,the overexpression of miR-21 by miR-21 mimic significantly abrogated metformin-mediated inhibition of the ability of HUVECs tubule formation.Conclusions: 1.The level of miR-21 in HUVECs was significantly downregulated by metformin in a dose-and time-dependent manner.2.Metformin treatment could significantly downregulate the level of miR-21 in HUVECs through the TGF-?1 pathway,and then significantly increase the expression levels of PTEN and SMAD7,inhibiting the phosphorylation of Akt,ERK1/2 and SMAD2/3.3.Metformin treatment could significantly downregulate the level of TGF-?1-induced miR-21 in HUVECs,and affect the corresponding target and related signaling pathway.Metiform exhibits the anti-angiogenesis through inhibiting ECs proliferation,migrantion and tubule formation.4.The overexpression of miR-21 abrogates the inhibitory effects of metformin treatment on the proliferation,migration,tube formation and protein-signaling phosphorylations of HUVECs.Our study broadens our understanding of the regulatory mechanism of miR-21 mediating metformin-induced anti-angiogenic effects,providing important implications regarding the design of novel miRNA-based therapeutic strategies against angiogenesis.