Inflammatory smooth muscle cell (iSMC) differentiation from residential vascular stem/progenitor cells (VSPCs) has been recently recognised as a critical determinant in cardiovascular diseases. We have recently reported an important role for microRNA-214 (miR-214) in mature vascular SMC functions and injury-induced neointima formation. However, little is known about the functional involvements of miR-214 in iSMC differentiation from VSPCs and its contribution to adverse arterial remodelling. In the current study, we aimed to study the functional importance of miR-214 and its target genes in iSMC differentiation and neointima SMC hyperplasia.Methods and results
miR-214 expression was significantly increased during SMC differentiation from VSPCs in response to TGF-β. miR-214 gain/loss-of-function assays showed that miR-214 plays an important role in SMC differentiation from VSPCs. By co-incubating VSPCs with TGF-β and TNFα, VSPCs were induced to differentiate toward iSMCs as evident by a decreased expression level of SMC-specific genes, but an increased level of inflammatory genes in iSMCs. Importantly, such phenotype could be inhibited/rescued by miR-214 over-expression. Both Suppressor of Fused (SuFu) and SMYD Family Member 5 (SMYD5, belongs to the class V-like SAM-binding methyltransferase superfamily) have been predicted as two of the top targets of miR-214 by several computational miRNA target prediction tools, and were indeed negatively regulated by miR-214 during iSMC differentiation. Luciferase assays showed miR-214 substantially repressed wild type, but not the miR-214 binding site mutated version of SuFu and SMYD5 (3’-UTR) gene reporter activity during iSMC differentiation, confirming both SuFu and SMYD5 are the functional targets of miR-214. Data from co-transfection experiments also revealed that inhibition of either SuFu or SMYD5 is required for the inhibitory effect of miR-214 on iSMC differentiation. Further mechanistic studies revealed that increased amount of the Glioma-Associated Oncogene Homolog 1 (GLI1) proteins were translocalized into nuclei in miR-214 over-expressing or SuFu knockdown cells, and the consensus sequence (GACCACCCA) of GLI1 within gene promoters of smooth muscle alpha-actin (SMαA) and serum response factor (SRF) was required for their regulation by miR-214 and SuFu. Importantly, locally enforced expression of miR-214 in the injured vessels significantly reduced SuFu and SMYD5 expression levels, inhibited iSMC number, and prevented neointima SMC hyperplasia after injury.Conclusions/implications
We have reported a novel role for miR-214 in iSMC differentiation from VSPCs and controlling neointima hyperplasia. Our findings provide new insights into the therapeutic effect of miR-214 in vascular diseases.