Mechanism Of CircDcbld1 In Regulating Intimal Hyperplasia Through MiR-145-3p/Neuropilictn-1 Pathway

Background and purpose: Angioplasty and stent implantation are important methods for the treatment of vascular obstructive diseases.Restenosis caused by intimal hyperplasia after surgery is an unavoidable problem after vascular reconstruction.Mechanisms such as vascular smooth muscle cells(VSMCs)hyperproliferation,migration,and extracellular matrix deposition are involved in the process of intimal hyperplasia.At present,drug-eluting balloon(DEB)and drug-eluting stents(DES)are used clinically to reduce vascular intimal hyperplasia.The main mechanism is the slow release of chemical drugs that inhibit cell proliferation and migration through the local slow release of the stent,thereby inhibiting the intimal hyperplasia of blood vessels.However,these drugs do not have specificity.While inhibiting VSMCs,they will also affect the function of vascular endothelial cells,delay the revascularization of blood vessels after vascular injury,increase the risk of thrombosis,and affect the prognosis of patients.Therefore,we need to further study the mechanism of vascular restenosis and try to find new ways to reduce vascular intimal hyperplasia.Recent studies have shown that noncoding RNA(ncRNA)is involved in regulating the pathophysiology of vascular injury.Circular RNA(circRNA)is a non-coding RNA made from linear RNA by reverse splicing.Because circRNA is a closed circular structure,it is not easily degraded by exonuclease,it is more stable than traditional linear RNA.circRNA regulates gene expression through a variety of ways,such as the adsorption of miRNA through a molecular sponge mechanism,the formation of complexes with RNA-binding proteins to regulate downstream signaling pathways,and even direct coding of proteins.Previous studies have shown that circRNA regulates the proliferation and migration of VSMCs.We examined the expression profiles of circRNAs of normal rat common carotid arteries(CCAs)and balloon-injured CCAs,and found that the expression of circDcbld1 significantly increased in balloon-injured CCAs.In this topic,we mainly explore the function and mechanism of circDcbld1 in balloon-injured vessels.The results of this research may provide a new target for reducing intimal hyperplasia after vascular reconstruction.Materials and methods: 1.The intimal hyperplasia of CCA of rats was injured by balloon,and an intimal hyperplasia model was established.Twelve-week-old male Sprague Dawley(SD)rats were used.Fogarty balloons injured the intima of the left common carotid artery,and the right CCA was used as a sham operation control.After 14 days,bilateral CCAs were taken for circRNA chip detection,and circRNAs with differential expression were screened and verified by PCR.2.Predict the downstream genes and biological signaling pathways of circDcbld1 through TargetScan and miRanda databases.3.Rat thoracic aorta VSMCs were cultured in vitro.SiRNA knocked down the expression of circDcbld1.Transwell chamber migration test,cell scratch test,and EdU infiltration test were used to detect the migration and proliferation of VSMCs;western blot was used to detect the levels of SM-MHC,α-SMA,and calponin of the contractile phenotype of VSMCs.4.Double luciferase reporter gene experiment verified the interaction of circDcbld1 with miR-145-3p and downstream targets;RNA fluorescence in situ hybridization(RNA-FISH)labeled circDcbld1,miR-145-3p in blood vessels Spatial location in tissues and cells;Western-blot and PCR explored the regulation of circDcbld1 on miR-145-3p/Neuropilin-1 signaling pathway.5.Copy the rat CCA intimal injury model,and inject chemically modified circDcbld1 siRNA around CCA.After 14 days,detect the circDcbld1 expression level and vascular intimal hyperplasia.Results: 1.The intimal hyperplasia of CCA was obvious after balloon injury,and the model was successfully established.2.Analysis of gene chip results showed that there were 73 circRNAs in the experimental group that had more than 2-fold difference in expression compared to the control group,38 expressions were up-regulated and 35 expressions were down-regulated(| FC |> 2,P <0.05).Among them,circDcbld1 was increased 7 times(n = 3,P <0.05).3.Transwell and cell scratch experiments confirmed that after circDcbld1 was silenced,the migration capacity of VSMCs decreased(n = 3,P <0.05),and this result could be reversed by miR-145-3p inhibitors;EdU results found that after circDcbld1 was silenced,there was no significant difference in the proliferation ability of VSMCs;Western blot results showed that the expression of contractile phenotype proteins(SMMHC,a-SMA,calponin)in VSMCs increased after silencing circDcbld1(n = 3,P <0.05).4.After silenced circDcbld1,the intimal hyperplasia caused by balloon injury was significantly reduced(n = 3,P <0.05).5.The results of the double luciferase reporter gene showed that there was an interaction between wild-type circDcbld1 and miR-145-3p.6.RNA-FISH experiments showed that both circDcbld1 and miR-145-3p were expressed in the cytoplasm of VSMCs,and their positions coincided(n = 4,P <0.05).7.Transfection of wild-type Neuropilin-1 reporter gene with miR-145-3p mimic into HEK293 T cells resulted in a significant decrease in luciferase activity.However,co-transfection of the mutant Neuropilin-1 reporter gene with miR-145-3p mimic did not significantly reduce luciferase activity.8.Western blot results showed that Neuropilin-1 expression decreased after circDcbld1 was knocked down(n = 6,P <0.05);Neuropilin-1 expression increased after using miR-145-3p inhibitor(n = 6,P <0.05)which can reverse the low expression of Neuropilin-1 caused by knocking down circDcbld1.Conclusions: 1.circDcbld1 is highly expressed in neonatal endometrium of rats;2.Silencing circDcbld1 reduces the migration ability of VSMCs,maintains the contractile phenotype,and significantly inhibits vascular intimal hyperplasia without significantly altering cell proliferation capacity;3.miR-145-3p is a downstream target gene of circDcbld1,circDcbld1 negatively regulates the activity of miR-145-3p;4.miR-145-3p negatively regulates the expression of Neuropilin-1;5.circDcbld1 through miR-145-3p/Neuropilin The-1 axis promotes the migration of VSMCs,leading to intimal hyperplasia.