MicroRNA-214 Regulates Smooth Muscle Cell Differentiation From Stem Cells By Targeting RNA-binding Protein QKI

Background and Objective:The basic pathological change of vascular injury lesions,is atherosclerosis,the basic pathological process is vascular repair and remodeling caused by vascular injury,followed by intimal thickening,leading to stenosis.Vascular smooth muscle cells are involved in the pathologic process by phenotypic transformation,proliferation,migration and differentiation as well as stem cells differentiation.Previous studies have found that microRNA-214(miR-214)could regulate the functional and phenotypic transformation of mature vascular smooth muscle cells.However,it remains unclear how miR-214 impacts stem cells differentiation into vascular smooth muscle cells.This study aims to reveal the role of miR-214 in the differentiation from embryonic stem cells into stem cells,identify the target genes of miR-214 and elucidate the mechanism.Methods:Established,stable,in vitro differentiation models of mouse embryo stem cells into smooth muscle cells were used to observe miR-214 expression,study its effect on embryonic stem cells differentiation into vascular smooth muscle cells.Secondly,bioinformatics analysis,luciferase gene reporter system and other methods were used to confirm the functional target genes of miR-214.Furthermore,co-immunoprecipitation(IP),dual luciferase gene reporter system and other molecular biological techniques were used to prove the effect and pathways of QKI on embryonic stem cells differentiation into vascular smooth muscle cells.Finally,in vitro transfected cells were injected into animals to simulate in vivo environment of mouse embryonic stem cell differentiation models in order to study the influence of miR-214 on embryonic stem cells differentiation into smooth muscle cells.Results:Mouse embryonic stem cells could differentiate into smooth muscle cells under the stimulation of Collagen IV by our previously established models and the expression of smooth muscle marker genes(SM-22a,SM-MHC,hl-Calponin)increased over time.MiR-214 expression was significantly increased during this process.In function experiments,over-expression of miR-214 could significantly promote the expression of vascular smooth muscle cell marker genes in mRNA and protein levels,while inhibition the expression of miR-214 decreased the expression of smooth muscle marker genes.A plurality of miRNA bioinformatics analysis databases predicted that QKI is one of the most likely target genes of miR-214.Overexpression of miR-214 could reduce QKI expression in mRNA and protein levels in the differentiating mouse embryonic stem cells while the inhibition of miR-214 could increase QKI expression levels.Luciferase gene reporter assay showed that overexpression of miR-214 could significantly reduce luciferase activity with QKI 3'UTR,while the inhibition of miR-214 could increase QKI 3'UTR luciferase activity to some extent.After the mutation of potential binding sites with miR-214 of QKI 3'UTR,the inhibition effect of miR-214 for luciferase activity disappeared.MiR-214 mimics and QKI plasmid co-transfection experiments showed that high expression of QKI could eliminate the promoting effect of overexpression of miR-214 on mouse embryonic stem cells differentiation into vascular smooth muscle cells.Meanwhile,overexpression QKI could reduce mRNA and protein levels of the vascular smooth muscle cell marker genes while the inhibition of QKI had the opposite effect by QKI overexpression and inhibition experiments.Furthermore,IP indicated that QKI could directly bind to the promoter sequence of the key transcription factors during the differentiation of smooth muscle cells.Finally,the in vivo differentiation models found that miR-214 could promote the expression of smooth muscle marker genes,which confirmed that miR-214 could promote the differentiation of mouse embryonic stem cells into vascular smooth muscle cells.Conclusion:This study successfully reveals the new features of miR-214 on embryonic stem cells differentiation into vascular smooth muscle cells.MiR-214 can promote differentiation of embryonic stem cells into vascular smooth muscle cells.QKI is the functional target gene of miR-214,which can be directly incorporated in the key transcription factor promoter region to inhibit differentiation.MiR-214 can directly bind to 3'UTR region of QKI mRNA to downregulate QKI expression,which influence stem cells differentiation into vascular smooth muscle cells.MiR-214 may be a new target on prevention and treatment of smooth muscle cell related vascular injury diseases.