The Mechanism Study Of LncRNA H19 Promoting Vascular Remodeling By Targeting Cyclin D1

Objective:With the advent of the aging population in our country and the improvement of people's living standards,the incidence of cardiovascular diseases is increasing year by year,and these diseases are mostly caused by vascular remodeling.Ischemic vascular disease associated with vascular stenosis caused by vascular remodeling is a serious threat to human health.At present,it is thought that the migration and proliferation of vascular smooth muscle cells are important factors leading to the vascular remodeling and atherosclerosis.In recent years,the studies of non-coding RNA in the field of vascular remodeling are more and more.Our previous work has demonstrated that LncRNA H19 can promote the proliferation of vascular smooth muscle cells.In 2013,it was found that LncRNA H19 is the ceRNA of let-7a.Cyclin D1 is an important protein that mediates cell proliferation by regulating the cell cycle.The aim of this study was to investigate whether LncRNA H19/let-7a/Cyclin D1 signaling pathway is involved in the regulation of vascular remodeling.Methods:In this study,rat models of carotid artery vascular remodeling were established,and the expression of Cyclin D1 in neointima was verified by immunohistochemistry.In order to investigate the function of let-7a in vascular smooth muscle cells,we up-regulated the expression of let-7a in vascular smooth muscle cells.Changes in cell proliferation were examined by CCK-8 assay and cell cycle distribution were determined by flow cytometry.The expression of Cyclin D1 was examined by qRT-PCR and Western Blot,and the binding of let-7a and Cyclin D1 was verified by luciferase assay.In order to study the function of LncRNA H19 in vascular smooth muscle cells,we up-regulated the expression of LncRNA H19 in vascular smooth muscle cells,and examined the changes of cell proliferation via CCK-8 assay,and determined the cell cycle distribution by cell flow.qRT-PCR and Western Blot were used to detect the expression of let-7a and Cyclin D1.LncRNA H19 expression was detected in let-7a up-regulated vascular smooth muscle cells.The binding sites and mfe of LncRNA H19 and let-7a were analyzed by DIANA software and RNAhybrid.GEO database was used to analyze whether the expression of let-7a in vascular remodeling was decreased.Finally,the binding relationship between LncRNA H19 and let-7a was verified by luciferase assay and RNA pull-down assay.Results:Immunohistochemistry showed that Cyclin D1 was highly expressed on the neointima of the vascular remodeling model.GEO database analysis showed that let-7a expression decreased in vascular remodeling.CCK-8 experiments showed that up-regulation of let-7a could inhibit VSMCs proliferation as well as up-regulation of LncRNA H19 could promote VSMCs proliferation.Flow cytometry analysis showed that cells overexpressing let-7a were mostly in the G1 phase of the cell cycle,and cells overexpressing LncRNA H19 were mostly in the S/G2 phase of the cell cycle.qRT-PCR and Western Blot experiments showed that up-regulation of let-7a could inhibit the expression of Cyclin D1,and upregulation of LncRNA H19 choud promote Cyclin D1 expression.qRT-PCR results showed that the expression of let-7a was decreased when H19 was overexpressed,while the expression of H19 was repressed when let-7a was overexpressed in VSMCs.The results of DIANA software and RNAhybrid analysis showed that LncRNA H19 and let-7a are highly likely to bind to each other.Luciferase assays and RNA pull-down assays further demonstrated that let-7a can target the 3'-UTR of Cyclin D1,and LncRNA H19 can sponge let-7aConclusion:The expression levels of cyclin D1 is up-regulated in neointima of balloon-injured artery.Let-7a has the function of inhibiting the proliferation of vascular smooth muscle cells.Let-7a directly targets on cyclin D1.LncRNA H19 can sponge let-7a.These results indicate that LncRNA H19 could down-regulate the expression of let-7a through competitive binding of let-7a,and further increase the expression of downstream target gene Cyclin D1 and promote the proliferation of vascular smooth muscle cells,resulting in the negative remodeling of blood vessels.