The Molecular Mechanism Of MicroRNA-206 Regulating The Smooth Muscle Cell Phenotype Conversion By Targeting ZFP580

Objective: It is new strategy to control the vascular smooth muscle cells(VSMC) phenotype conversion in order to prevent restenosis after PCI. As the regulators of VSMC phenotype conversion, Micro RNAs can influence the phenotype conversion of VSMC by down-regulation its downsteeam target genes expression at the post-transcriptional levels. ZFP580, a novel C2H2 type zinc finger transcription factor cloned by our lab, could modulate the vascular remodel by means of regulating the proliferation and migration of endothelial cells and VSMC. It was newly confirmed by our lab that miccro RNA-206 can down-regulate the expression of ZFP580 and promote the conversion of VSMC from contractile to synthetic type. Our project will plan to use lenti virus vector to micro RNA-up/ micro RNA-down the micro RNA-206 of primary VSMC in vitro. Furthermore, we identified the molecular mechanism of Micro RNA-206 regulating the smooth muscle cell phenotype conversion by targeting ZFP580. Our study will provide new scientific data to prevent restenosis after PCI.Methods:Extraction and separation of rat VSMC,Bioinformatics analysis of the target gene binding site for mi R-206 and ZFP580. Identified by morphology and immunohistochemistry. Use the TGF-β(2ng/ml) stimulated VSMC in different time to find the best time point. And control group were treated with equal amounts of DMEM culture medium. The expression levels of mi R-206 was determined by Realtime-PCR. Western-blot detected the protein expression levels of ZFP580、SMa-actin, SM22 a, Smad2/3, phospho-Smad2/3. Use lenti virus vector to micro RNA-up/ micro RNA-down the micro RNA-206 of primary VSMC or use SB431542 inhibitors in vitro, the detecting the related index. Luciferase was be used to identified the interrelation of mi R-206 and ZFP580.Results:(1) In our studies(and in agreement with the work of others), we applied a well-established VSMC model for phenotypic modulation in which VSMC differentiation was induced by Transforming Growth Factor-beta1(TGF-β1). Control group were treated with equal amounts of DMEM culture medium. Using cultured rat VSMCs, we first examined the effect of TGF-β1 on the expression of two VSMC differentiation markers such as SM22α and SMα-actin. TGF-β1(2ng/ml) increased expression of SM22α, phospho-Smad 2/3 and SMα-actin in a time- and dose-dependent Manner(P<0.05). But the expression of Smad2/3 had no significant difference. Interestingly, the opposited expression pattern was found in mi R-206 levels in VSMCs after TGF-β1 stimulation. TGF-β1(2ng/ml) caused a rapid decrease in mi R-206 expression at 12 h.(P<0.05), as demonstrated by q RT-PCR. ZFP580 expression was indeed increased in TGF-β1-stimulated VSMCs, reaching a maximum at 12h(P<0.05).(2) To determine the role of TGF-β1/smad in the down-regulation of mi R-206 in VSMCs, SB431542, a selective cell-permeable inhibitor of TGF-β1/smad, is widely used to inhibit Smad2/Smad3 activation, The efficacy of SB431542 was confirmed by its ability to abolish TGF-β1-induced phosphorylation of Smad2/3. Normal VSMC or Pretreatment of VSMCs with SB431542(20um) or DMSO and after TGF-β1(2ng/ml) stimulation for 12 h significantly inhibited the TGF-β1-mediated down-regulation of mi R-206 compared with the DMSO or Normal groud(P<0.05). the opposited protein expression levels of ZFP580 was found in VSMCs, significantly inhibited the TGF-β1-mediated up-regulation of ZFP580(P<0.05). The similar expression pattern was found in SM22α, SMα-actin and phospho-Smad2/3 levels in VSMCs(P<0.05)(3) In our recent study, TGF-β1 can significantly decrease the expression levels of mi R-206.To overexpress the expression of mi R-206 in VSMCs, Lv-rno-mi R-206 was used. At 12 h after infection, mi R-206 expression was increased significantly in Lv-rno-mi R-206-treated VSMCs compared with that in VSMCs treated with LV-GFP with or without TGF-β1(P<0.05). However, Lv-rno-mi R-206 inhibited the TGF-β1-mediated effects on VSMC differentiation as shown by the expression changes of VSMC differentiation marker genes such as SM22α and SMα-actin were downregulated at protein levels(P<0.05). These results indicate that TGF-β1-mediated VSMCs differentiation was affected by the mi R-206.(4) If ZFP580 is a target gene for mi R-206, its expression should be upregulated in cultured differentiated VSMCs stimulated with TGF-β1, because mi R-206 expression is downregulated by TGF-β1. ZFP580 expression was indeed increased in TGF-β1-stimulated VSMCs. To confirm that ZFP580 is a target gene of mi R-206 in cultured VSMCs, both gain-of-function and loss-of-function approaches were applied. As expected, ZFP580 was up-regulated by mi R-206-inhibitor(P<0.05) and down-regulated by Ad-mi R-206(P<0.05) at the protein level.(5) To determine the role of ZFP580 in the phenotype switching of VSMCs. both gain-of-function and loss-of-function approaches were applied. As expected, SMa-actin、SM22a was up-regulated by Lenti-ZFP580(P<0.05) and down-regulated by i RNA-ZFP580(P<0.05) at the protein level.(6) Luciferase activity analysis: 293 T cells transfect with Ad-mi R-206 or Ad-GFP. Production of Luc-3’ UTR reporter constructs of ZFP580 and assay for mi R-206 mediated reduction in ZFP580. The luciferase(luc) reporter construct consists of a strong CMV promoter that drives luc expression. 293 T cells cotransfect with p GL-3M-zfp580 3’UTR、p RL-CMV plasmid. Luciferase activity were normalized to that of p RL-CMV plasmid. And fold induction calculated with respect with control. These results indicate that overexpress mi R-206 can inhibited the Luciferase activity of ZFP580.Conclusion:TGF-β can inducate the VSMC phenotype switching. upregulated the expression of differentiation mark such as SM22α and SMα-actin in VSMC. TGF-β regulate the expression of mi R-206 and ZFP580 through the Smad2/3 pathway. Overexpress the mi R-206 could downregulate the expression of ZFP580, SM22α and SMα-actin, inhibited the VSMC differentiation.