Linc00665-207 Functions As A Competing Endogenous RNA To Regulate CCND1 Expression In Nicotine-induced Excessive Proliferation Of Vascular Smooth Muscle Cells

Objective:The prevalence of peripheral artery stenosis is about 4.3%(ABI<0.9 of any lower limb)in people more than 40y,and the prevalence of people more than 70y is 14.5%.70%of patients with severe ischemia of lower limb obtain vascular reconstruction,75%of them ameliorate their symptom.However,the rate of amputation and death are still 21.5%and 13.5%respectively.Smoking is a more important risk factor of peripheral artery disease than hypertension and diabetes mellitus.The risk of peripheral artery disease of smokers is 4.46-fold than that of non-smokers.In terms of the already-patient with peripheral artery disease,to refuse to quit smoking often brings a development into severe ischemia of lower limb,which needs the amputation or vascular reconstruction.It is the key point of occurrence and recurrence of vascular stenosis that anormal cell cycle trigger the excessive proliferation of vascular smooth muscle cells(HA-VSMC).Nicotine,as the major toxicological component in tobacco,participates in the promotion of vascular smooth muscle cell proliferation.But whether nicotine promote vascular smooth cell proliferation via regulating cell cycle is not clear.Non-coding RNA plays important roles in vascular stenosis.How does non-coding RNA regulate vascular smooth muscle cell proliferation and is it associated with cell cycle regulation?Our previous study has suggested that CCND is associated with G1/S phase switch and could regulate vascular smooth muscle cell proliferation.MicroRNA hsa-let-7a-5p expresses lower in both stenotic artery and vein than that of normal vessels.The bioinformatics prediction found that there was a underlying competing endogenous RNA(ceRNA)pattern among long non-coding RNA linc00665-207,hsa-let-7a-5p and CCND1.Whether long non-coding RNA(lncRNA)is involved in the process that nicotine promotes vascular stenosis is not clear,nevertheless,the bioinformatics prediction suggested that RelA could regulate linc00665-207.Therefore,we tried to explore whether linc00665-207 participate in the process that nicotine promotes vascular smooth muscle cell proliferation and the underlying mechanism.Methods:In current study,CCK-8 and flow cytometry were used to assess proliferation and apoptosis of vascular smooth muscle cells.Scratch test was used to assess migration of vascular smooth muscle cells.Real-time PCR and western blotting were used to assess gene expression.Immunocytochemistry was used for detection of RelA translocation ChIP assay and luciferase assay were used to determine the molecular interaction in vascular smooth muscle cellsResults:Nicotine showed a more powerful proliferation promoting action in vascular smooth muscle cells than platelet derivative growth factor(PDGF-BB).Besides,linc00665-207 showed no difference between PDGF-BB-treated group and control group,but an obvious upregulation of linc00665-207 was observed in nicotine-treated group compared with control group.What’s more,merely overexpressing RelA could not affect proliferation and expression of linc00665-207,hsa-let-7a-5p,CCND1/2 in vascular smooth muscle cells.The further study found that nicotine could promote RelA expression,activation and nuclear translocation.Nicotine could activate the overexpression RelA compared with the overexpressed group without nicotine stimulation,then upregulate the expression of linc00665-207 and CCND 1/2,downregulate the expression of hsa-let-7a-5p,promote proliferation and migration of vascular smooth muscle cells and reduce the apoptosis.Silence of linc00665-207 could reserve partly the CCND1 expression but not affect the expression of CCND2 and the proliferation,migration and apoptosis of vascular smooth muscle cells.Overexpression of linc0065-207 could downregulate the expression of hsa-let-7a-5p,upregulate expression of CCND1,but could not affect CCND2 expression and migration and apoptosis of vascular smooth muscle cells,which could be all reversed by hsa-let-7a-5p overexpression.Dual luciferase reporter assay showed that there was a specific binding between RelA and promoter of linc0065-207.Chromosome immunoprecipitation suggested that nicotine could promote the interaction between RelA and promoter of linc0065-207.RNA binding protein immunoprecipitation assay confirmed that there was a direct interaction between linc00665-207 and hsa-let-7a-5p.Dual luciferase reporter assay after a mutation of the predicted binding sites on by one suggested that the NO.1920-1937 nucleotides of linc00665-207 was the only one effective binding site with hsa-let-7a-5pConclusion:Nicotine-activated RelA promotes the expression of linc00665-207,which then upregulates CCND1 by sponging hsa-let-7a-5p.Our work has demonstrated for the first time that a lncRNA is associated with nicotine-related HA-VSMC excessive proliferation.It could explain excessive HA-VSMC proliferation in smoking-related vascular stenosis,and facilitate the development of novel diagnostic and therapeutic strategies.