| BackgroundAtherosclerosis is an inflammatory disease of the vascular arteries,which eventually forms arterial plaque that continuously results in plaque hardening and blood vessels thickening.These plaques are composed of immune cells,mesenchymal cells,lipids,and extracellular matrix.Current researches mainly aimed to explore the source of mesenchymal cells of the plaque,and these studies indicate that one of their sources is the activation and proliferation of quiescent fibroblasts or the phenotypic transition of epithelial cells(ie,Epithelial-mesenchymal transition),while recent studies showed that another source of mesenchymal cells was endothelial mesenchymal transition.Endothelial-to-mesenchymal transition(End MT)means that endothelial cells lose cellto-cell connections mediated by membrane proteins,including VE-cadherin and CD31,resulting in endothelial cell polarity changes,and enhanced ability of migration and invasion.Additionally,endothelial cells acquire a mesenchymal phenotype,and the increased expression of mesenchymal or myofibroblast markers(such as vimentin andα-smooth muscle actin)causes endothelial cells to stratify and detach from endothelial layer.Several studies have reported that End MT is regulated by various of stimulating factors,among them transforming growth factor 1(TGF-β1)is considered the driving force of End MT.There are two main pathways in End MT,induced by TGF-β1.1.Classical Smad-dependent pathway: Smad family proteins as intracellular signaling mediators.2.Smad-independent pathways: multiple non-Smad signaling pathways mediate End MT via regulating TGF-β / Smad signaling(ie.AMPK,NF-κB signaling).Smad ubiquitin regulator 2(Smurf2)is an E3 ubiquitin ligase and a negative regulator of Smad2,which could regulate TGF-β / Smad signaling by mediating the degradation of Smad2.RNA-binding proteins(RBPs)play an important role in various pathophysiological processes,including myocardial ischemia,fibrosis,angiogenesis,cell death,proliferation,and cellular metabolism.RBPs act at various steps in the posttranscriptional regulation including m RNA splicing,processing,transport and localization.HuR as one of acknowledged RBPs,leads to stabilization or destabilization of the target transcripts by specifically binding to ARE sequence elements on the 3′-UTR and / or 5′-UTR region of the m RNA.The studies reported that HuR mainly regulated target genes through the following mechanisms: 1.binding directly to ARE elements of m RNA,2.binding to mi RNA or other non-coding RNA,3.acting at the epigenetic level through m6 A to stabilize the target transcripts,and 4.be involved in the export of target m RNA to cytoplasm stabilize.According to biological information analysis,HuR may combined with Smurf2 m RNA.We speculated that Smurf2 may be regulated by HuR,thereby participating in the process of End MT.Therefore,we propose hypothesis that TGF-β1 may inhibit the expression of Smurf2 by regulating the formation of HuR / Smurf2 m RNA complex,thereby inhibit the degradation of phosphorylate Smad2 and activate the End MT.ObjectivesThis study explores whether TGF-β1 could regulate the Smad2-mediated End MT by regulating HuR / Smurf2 m RNA complex.Methods1.TGF-β1 induces End MT in HUVECs.(1)After treatment of HUVECs with 10ng/ml TGF-β1 for 24 h and 48 h,the expression of End MT-related protein was detected by Western Blot;after treatment with TGF-β1 at 10ng/ml for 24 h,the migration ability of HUVECs’ were tested by Wound Healing and Transwell assay.(2)After treating HUVECs with TGF-β1 10ng/ml for 24 h,the m RNA and protein levels of Smurf2 were detected by PCR and Western Blot respectively.Furthermore,Smurf2 was overexpressed and the expression level of p-Smad2 was examined.(3)HUVECs were treated with TGF-β1 10ng/ml for 24 hours,and the treatment group and control group were treated with 5μg/m L actinomycin D for 2h,4h,6h,8h,and 12 h,respectively.PCR and RT-q PCR were used to detect the degradation of Smurf2 m RNA.2.To investigate the effect of HuR on the stability of Smurf2 m RNA(1)We used biological information to analyze the binding of HuR to Smurf2,and verified the binding of HuR to Smurf2 m RNA by m RNP experiment.(2)We tested HuR the m RNA and protein levels of Smurf2 and m RNA stability after HuR KD using si RNA.And we were examined the migration of HUVECs using Transwell.(3)Following overexpression of HuR using adenovirus,we detected the m RNA and protein levels of Smurf2 and m RNA stability,and the migration of HUVECs by Wound Healing and Transwell assay.3.To explore the effect of TGF-β1 on the End MT induced by the formation of HuR / Smurf2 m RNA complex.(1)After treating HUVECs with TGF-β1 10ng/ml for 24 h,we tested the formation of HuR / Smurf2 m RNA complex.(2)After HuR overexpression treatment for 48 h,and stimulation with TGF-β1 at10ng/ml for 24 hours,PCR and Western Blot were used to detect the levels of Smurf2 m RNA and protein.(3)After HuR overexpression treatment for 48 h,and stimulation with TGF-β1 at10ng/ml for 24 hours,migration of HUVECs was tested by Wound Healing and Transwell assay.Results1.TGF-β1 promotes p-Smad2-induced End MT by inhibiting the expression of Smurf2,which enhances the migration of HUVECs,the difference is statistically significant(P<0.05);2.Biological information analysis and m RNP experiments verified that Smurf2 m RNA has HuR-binding;increased degradation of Smurf2 m RNA,decreased Smurf2 protein expression and enhanced migration ability of HUVECs were observed after HuR knockdown,and the opposite results were found after HuR overexpression(P<0.05);3.TGF-β1 inhibits the formation of HuR / Smurf2 m RNA complex and accelerates the degradation of Smurf2 m RNA;under the stimulation of TGF-β1,overexpressed could promotes Smurf2 expression,thereby depresses the TGF-β induced phosphorylation of Smad2,and the End MT(P<0.05).ConclusionsOur data indicated that TGF-β1 inhibited the binding of HuR to Smurf2 m RNA,enhanced the degradation of Smurf2 m RNA,and activates Smad2-mediated End MT. |
PDF Full Text Request