MicroRNA-31Controls Phenotypic Modulation Of Human Vascular Smooth Muscle Cells By Regulating Its Target Gene Cellular Repressor Of E1A-stimulated Genes

Objective Phenotypic modulation of vascular smooth muscle cells (VSMCs) plays acritical role in the pathogenesis of a variety of proliferative vascular diseases. Wepreviously determined that the cellular repressor of E1A-stimulated genes (CREG) playsan important role in phenotypic modulation of VSMCs, but the mechanism of its upstreamsignaling regulation is not clear. Recently, microRNAs (miRNA) have been found to playa critical role in cell differentiation and proliferation via regulating its target gene.Computational analysis shows many miRNAs are able to bind to CREG mRNA3’untranslated region (3’-UTR), suggesting that miRNA may be an upstream regulator ofCREG. Thus, which miRNA bind to CREG directly and involved in CREG-mediatedeffect on VSMCs phenotypic modulation is an important question worth to investigate. Methods Human VSMCs were stimulated by platelet derived growth factor (PDGF)with different concentrations and serum starvation (0.1%) to establish phenotypicmodulation model. Expression of CREG and VSMC differentiation marker genes, SMα-actin and calponin, in different phenotypic VSMCs were determined by Western blot.Computational analysis has suggested that miR-31is able to bind to CREG mRNA3’-UTR. To find the miRNA which has a negative relationship with CREG, CREG andmiR-31expressions were determined by qRT-PCR in VSMCs with different phenotype.Then we use miRNA mimic and inhibitor to overexpress and knockdown miRNAexpression, and analyze SM α-actin and CREG expressions by Western blot and qRT-PCR.To identify miR-31can bind to CREG directly, luciferase expression of a firefly luciferasereporter construct containing CREG mRNA3’-UTR was measured on a scintillationcounter by using a dual luciferase reporter system. Additionally, CREG was knockeddown by its shRNA in VSMCs and miRNA inhibitor was transfected into CREG deficientcells, SM α-actin expression were determined by Western blot. At last, we determinedmiR-31levels in the serum of63patients coronary artery disease (CAD) and in37healthycontrols. Patients with CAD consisted of32CAD subjects with first-episode restenosisand31CAD patients without restenosis.Results In cultured VSMCs, VSMC differentiation marker genes and CREGexpressions were downregulated in quiescent differentiated VSMCs and upregulated inproliferative cells treated by PDGF and serum starvation. As expected, miR-31and CREGhave a negative relationship at protein and mRNA level in VSMCs with differentphenotype. Furthermore, gain-of-function and loss-of-function showed that SM α-actinand CREG expressions are suppressed by miR-31mimic and are increased by miR-31inhibitor in vitro. More importantly, miR-31mimic decreased luciferase expression drivenby the construct of CREG mRNA3’-UTR containing the miR-31binding site in HEK293cells, confirming CREG is a direct target of miR-31. Furthermore, knockdown of CREGin cultured VSMCs, the effect of miR-31inhibitor on SM α-actin expression is decreasedin CREG deficient cells. Finally, miR-31levels were higher in the serum of CAD patientswith restenosis compared to CAD patients without restenosis and healthy controls.Conclusion We conclude that miR-31not only directly binds to its target gene CREGand modulates VSMC phenotype through this interaction, but also can be an importantbiomarker in diseases involving VSMC phenotypic modulation. These novel findings mayhave extensive implications for the diagnosis and therapy of a variety of proliferative vascular diseases.