The Role Of LncRNA PEBP1P2 In Vascular Smooth Muscle Cell Proliferation And Phenotypic Switching

Objective: To investigate the role of long non-coding RNAs(lncRNAs)in regulating the proliferation,migration and phenotypic switching and its mechanism in vascular smooth muscle cells(VSMCs).To explore the intervention and precise target for cardiovascular disease(CVD).Methods:(1)The new long non-coding RNA PEBP1P2 was obtained from the data,and its protein-coding ability was predicted by bioinformatics method.(2)The expression level of the PEBP1P2 in different cell lines,serum of coronary heart disease patients,and atherosclerotic plaques were evaluated by qRT-PCR.(3)The small interfering RNA of PEBP1P2 was designed and synthesized,and its knockdown efficiency in VSMCs were verified by qRT-PCR.After PEBP1P2 knockdown using siRNA in VSMCs,the cell proliferation was detected by EdU and CCK-8,cell migration was determined by wound?healing and transwell,and the expression level of contraction genes and CCND1 was evaluated via qRT-PCR and western blotting.(4)The PEBP1P2 overexpression vector was prepared,and the overexpression efficiency in VSMCs were detected by qRT-PCR.After PEBP1P2 overexpression in VSMCs,the cell proliferation was detected by EdU and CCK-8,cell migration was determined by wound?healing and transwell,and the expression level of contraction genes and CCND1 was evaluated via qRT-PCR and western blotting.(5)The expression of PEBP1P2 in VSMCs induced by PDGF-BB was detected using qRTPCR.The EdU,cck-8,wound?healing,transwell,and western blotting were used to detect the effect of PEBP1P2 overexpression on VSMCs stimulated by PDGF-BB.(6)qRT-PCR was used to determine the expression level of PEBP1P2 neighbor genes after PEBP1P2 knockdown or overexpression in VSMCs.(7)The bioinformatics method was used to predict the downstream binding protein of PEBP1P2 and its binding capacity to the binding protein.The RNA Immunoprecipitation assay was used to further verify the prediction results.The expression of the downstream binding protein CDK9 after PEBP1P2 knockdown or overexpression was detected by western blotting.(8)The small interfering RNA of CDK9 was designed and synthesized,and its knockdown efficiency in VSMCs were verified by qRT-PCR and western blotting.The CCK-8,wound?healing,transwell,and western blotting were used to evaluate the biological function of VSMCs after CDK9 knockdown or both PEBP1P2 and CDK9 knockdown.(9)The expression of CDK9 in VSMCs induced by PDGF-BB was detected using western blotting.The cck-8,wound?healing,and transwell,were used to detect the effect of CDK9 knockdown on VSMCs stimulated by PDGF-BB.(10)The CDK9 overexpression vector was constructed and its overexpression efficiency in VSMCs verified by western blotting.The CCK-8,wound?healing,transwell,and western blotting were used to evaluate the biological role of VSMCs after CDK9 overexpression or both PEBP1P2 and CDK9 overexpression.Results:(1)PEBP1P2 was enriched in VSMCs,and down-regulated in blood sample of coronary heart disease patients and atherosclerotic plaques.(2)The proliferation,migration and phenotypic switching of VSMCs were significantly enhanced after PEBP1P2 knockdown.In contrast,Overexpression of PEBP1P2 could significantly inhibit VSMCs proliferation,migration and phenotype switching.In addition,overexpression of PEBP1P2 can observably restrain the proliferation,migration,and phenotypic switching of VSMCs induced by PDGF-BB.(3)PEBP1P2 plays a regulatory role by directly binding to CDK9.(4)The proliferation,migration and phenotype conversion of VSMCs were markedly suppressed after CDK9 knockdown;In contrast,CDK9 overexpression dramatically improved the proliferation,migration and phenotype conversion of VSMCs;and the above process could be reversed by PEBP1P2 knockdown or overexpression.Conclusion: We identified a novel lncRNA-PEBP1P2,which plays an important role in VSMCs proliferation,migration,and phenotypic transformation by directly binding to CDK9,and explored its clinical significance.This study enriched the functional regulation and mechanism of VSMCs,and provided a new perspective and target for the diagnosis and treatment of CVD.