Circular RNA WDR77 Regulates Vascular Smooth Muscle Cells Proliferation And Migration Via MiR-124/FGF-2

Background and aimsAcute coronary event(unstable angina pectoris,acute myocardial infarction)is a common and major disease that endangers human health.Acute coronary events are caused by the rupture of atherosclerotic plaque in the coronary arteries and secondary thrombosis.Many factors are involved in the development of atherosclerotic lesions.Smooth muscle cell dysfrnction plays an important role in intimal hyperplasia,plaque formation and vascular stenosis.Vascular smooth muscle cells(VSMCs)is the main component of vascular wall cells.VSMC is not a terminal differentiated cell,and it has high plasticity.When the blood vessels are damaged,VSMCs dedifferentiated and transformed from a contractile type to a synthetic type to promote vascular repair.Once the damage was repaired,the VSMCs of the healthy blood vessels changed back to the non-proliferative systolic phenotype.But in pathological conditions,the dysfrnction of vascular smooth muscle cell phenotype will lead to the development of cardiovascular disease.Abnormal VSMCs phenotype plays an important role in the development of atherosclerosis and restenosis after angioplasty.Recently,some scholars have obtained a new class of stem cells in the arterial wall,the expression of Sox 17,Sox 10 and S100 P pluripotent markers that can differentiate into smooth muscle cells;the study found that in part after vascular injury,synthetic VSMCs does not come from the contraction type VSMCs to differentiation,but from pluripotent the blood stem cells,suggesting that vascular pluripotent stem cells to differentiate into type VSMCs synthesis is an important mechanism in the pathogenesis of atherosclerotic disease.Many studies have shown that the pathological process of heart is often accompanied by the alteration of gene expression profile of cardiac function.With the deepening of basic research,more and more studies have found the important role of circular RNA in cardiovascular diseases.However,the expression and regulation mechanism of circulating RNA in vascular smooth muscle cells(VSMCs)are not clear.In this study,the microarray analysis of circular RNA was used to explore the expression profiles of circular RNA in smooth muscle cells induced by high glucose in vitro.We applied the downstream targets of biological information to explore the regulation of circular RNA,and through the function test to further verify the mechanism of the proliferation and migration of circular RNA and a downstream target of the signaling pathway on vascular smooth muscle cells.This study provides a new possible theoretical basis for circular RNA related vascular lesions.Materials and methods1.Microarray analysis was used to analyze circulating RNA to explore the expression of circular RNA in smooth muscle cells induced by high glucose in vitro.2.The human vascular smooth muscle cells were cultured in vitro,and the circWDR77 gene was knocked out by siRNA.We detected the number of surviving cells in vascular smooth muscle cells by MTT,and used flow cytometry to analyze the cell cycle.In addition,we also conducted the Transwell Migration Experiment and the Healing Test to explore the effect of circWDR77 on the proliferation and migration of vascular smooth muscle cells.3.The bioinformatics prediction method was used to analyze the downstream target of circWDR77.We used luciferase determination and RNA immunoprecipitation to investigate the effect of circWDR77 on the miR-124 expression.4.We used the Western blot and RT-PCR to further verify and clarify the regulatory role of circWDR77 on miR-124/FGF2 signaling pathway and possible mechanisms.Results1.Our team performed high glucose(27.5 mmol/L)and normal glucose(5.5 mmol/L)treatment to simulate diabetic condition in vitro.After normalization,total 983 circRNAs were dysregulated,including 458 up-regulated and 525 down-regulated.Among these different expression circRNAs,31 circRNAs were up-regulated and 22 circRNAs were down-regulated with 2 folds change(P<0.05).2.Expression of circWDR77 in high glucose treatment VSMCs was significantly up-regulated compared to normal group.3.Transfected with si-circWDR77,expression of circWDR77 was significantly decreased in high glucose induced VSMCs.MTT assay showed that circWDR77 silencing could decrease the living VSMCs number compared to control group.Cycle analysis detected by flow cytometry showed that circWDR77 silencing induced G0/G1 phase arrest and suppressed cycle progression.Transwell migration and wound healing assay showed that circWDR77 silencing decreased the migrated cells and distance compared to control group.4.Luciferase reporter assay,RIP assay and RT-PCR showed that miR-124 was significantly down-regulated in VSMCs treated with high glucose compared to that in normal glucose.5.Expression of FGF2 mRNA and protein showed that circWDR77 silencing decreased FGF2 expression,which was reversed by miR-124 inhibitor.MTT assay showed the reversion of miR-124 towards circWDR77 on proliferation.Cycle analysis revealed that miR-124 inhibitor recovered the GO/G1 phase arrest induced by circWDR77 silencing.Transwell and wound healing assay showed that miR-124 inhibitor rescued the migration inhibition causedby circWDR77 silencing.Conclusions1.The increase of blood glucose can cause significant differential expression of circRNAs in vascular smooth muscle cells.2.Expression of circWDR77 in high glucose treatment VSMCs was significantly up-regulated compared to normal group.3.The circWDR77 silencing could inhibit the high glucose induced VSMCs proliferation and migration.4.The circWDR77 targeted tomiR-124 and represented opposite expression,suggesting the molecular sponge role of circWDR77 on miR-124.5.The results indicating the molecular sponge role of circWDR77 on miR-124 via targeting FGF2,regulating the proliferation and migration of VSMCs.6.The above pathway is an effective downstream regulation of circWDR77 on VSMCs proliferation and migration.