| INTRODUCTION: Pulmonary hypertension is a kind of fatal disorder characterized by persistent elevation of resistance and pressure in pulmonary artery,with decreased activity tolerance,increased right ventricular load and severe right heart failure as major clinical symptom.Pulmonary arterial hypertension(PAH)is the most detrimental type,involving extensive remodeling of the pulmonary arteries mediated by hyperproliferation and migration of pulmonary arterial smooth muscle cell(PASMC).The long non-coding RNA(lncRNA)is a recently discovered regulator involved in kinds of diseases,However,little is known about the role of lncRNA in pulmonary arterial remodeling and PAH development.OBJECTIVE: To screen vascular specific lncRNAs and study their expression pattern,functional role and molecular mechanism during pulmonary arterial remodeling and PAH development.METHODS AND RESULTS: A group of lncRNAs regulated by PDGF-BB were identified with RNA sequencing,and one of these lncRNAs was found to be expressed specifically with high level in vascular tissue via qRT-PCR,moreover,this lncRNA was significantly downregulated in pulmonary arteries of PAH rats induced by monocrotaline or hypoxia,hence named PAH Associated Vascular Specific LncRNA-1(PAVSL1).The gene structure of PAVSL1 was clarified through rapid-amplification of cDNA ends,and the predicted promoter region of PAVSL1 was proved with high transcriptional activity by dual luciferase reporter assay.PDGF-BB downregulated PAVSL1 in a time-and dose-dependent manner,however,PDGF-BB did not affect the transcriptional activity of PAVSL1 significantly.In addition,downregulation of PAVSL1 induced by PDGF-BB was fully abrogated when phosphatidylinositol 3'kinase(PI3K),protein kinase C(PKC)and extracellular regulated protein kinases(ERK)were inhibited synchronously with their corresponding inhibitors(Pctilisib,Enzastaurin and U0126-EtOH),indicating that PAVSL1 was downregulated by PDGF-BB via PI3 K,PKC and ERK pathways together.To investigate the functional role of PAVSL1 in PASMC,RNA sequencing was performed to identify the differentially expressed genes after PAVSL1 was knocked down with shRNA.GO(Gene Oncology)and KEGG pathway analysis prompted that PAVSL1 might involve in the migration of PASMC,indeed,the result of wound-healing assay proved that loss of PAVSL1 promoted the migration of PASMC,and the mobility of PASMC was suggested to be increased by knockdown of PAVSL1 through single cell motion tracer assay.The expression of MMP2,which is related to migration of PASMC,was also upregulated by PAVSL1 knockdown.Moreover,immunofluorescence staining showed that loss of PAVSL1 promoted the extension of microtubule,localization of microtubule organization center(MTOC)in the orientation of migration,and the construction of focal adhesion during migration.To explore how PAVSL1 regulated migration of PASMC,bioinformatics analysis was further performed with the differentially expressed genes,and 39 genes of the top 60 downregulated genes were classified into 16 gene clusters according to their location on genome,indicating that PAVSL1 modulated the chromatin structure.Additionally,sub-fraction and RNA fluorescence in situ hybridization(FISH)proved that PAVSL1 mainly located in the nucleus.Furthermore,loss of PAVSL1 significantly increased the methylation level of the twenty-seventh lysine in histone 3(H3K27)and the thirty-sixth lysine in histone 3(H3K36).CONCLUSION: In summary,this study identified a novel vessel specific lncRNA PAVSL1,which was downregulated by PDGF-BB via PI3 K,PKC and ERK pathways together.Inhibition of PAVSL1 expression increased the methylation level of H3K27 and H3K36,suppressing the expression of downstream targets epigenetically and promoting the migration of PASMC through mediating microtubule extension,direction of MTOC and construction of focal adhesion.These data provide novel insights into the role of lncRNA PAVSL1 in the modulation of PASMC migration,vascular remoldeling and PAH development. |
PDF Full Text Request