The Role And Mechanism Of CircGSAP Regulating Pulmonary Vascular Remodeling In Pulmonary Hypertension Via MiR-27a-3p/BMPR2 Signaling Pathway

BackgroundPulmonary hypertension(PH)is a serious disease,which has a variety of clinical manifestations and makes most cardiovascular and respiratory diseases complication.PH has been considered as a serious threat to human physical and mental health.The major pathological changes of PH are pulmonary vascular remodeling,which is characterized by the accumulation of different vascular cells(pulmonary artery endothelial cells(PAECs),smooth muscle cells,fibroblasts,myofibroblasts and pericytes).It is accompanied by infiltration.Although some targeted drugs have been used to treat PH,these drugs are designed to dilate pulmonary vessels and cannot completely reverse pulmonary vascular remodeling and eliminate inflammation.Therefore,in order to reverse pulmonary vascular remodeling,it is urgent for us to better understand the pathophysiological mechanism of pulmonary artery remodeling.Pulmonary vascular endothelial cells are a layer of flat epithelium lining the inner surface of pulmonary blood vessels,which form the inner wall of blood vessels.PAECs play an indispensable role in maintaining vascular homeostasis.For PH,PAECs damage is the primary factor in pulmonary vascular remodeling.External stimulation,such as long-term hypoxia,will cause dysfunction of PAECs,and eventually lead to excessive proliferation,excessive migration and apoptosis resistance.These changes disrupt the homeostasis of pulmonary angiogenesis and repair mechanisms,ultimately leading to pulmonary vascular remodeling.Therefore,it is important to correct the dysfunction of PAECs to solve PH pulmonary vascular remodeling.Circ RNAs are non-coding RNAs widely present in mammalian cells.Their heads and tails are spliced to form covalently closed structures.It is characterized by abundant,stable,diverse and conserved expression,as well as species-specific,and has many important regulatory functions at the post-transcriptional level.More and more evidences indicate that circ RNAs are involved in the occurrence and development of PH.However,at present,only circ RNAs in plasma are used to evaluate the prognosis of idiopathic pulmonary hypertension(IPAH),and there is no study on the differential expression of circ RNAs in peripheral blood mononuclear cells(PBMCs)to evaluate the prognosis of PH.In addition,there are only reports of circ RNAs in pulmonary vascular smooth muscle cells,and few studies focus on the effect of circ RNAs on the PAECs.PAECs dysfunction is the first factor of pulmonary vascular remodeling.It is particularly important to explore the mechanism of circ RNAs in pulmonary vascular remodeling and PAECs dysfunction.ObjectiveThe purpose of this study is to find new circ RNAs,explore the meaningful of circ RNAs on the diagnosis and prognosis of PH,and further explore the influence of circ RNAs on the function of PH under normal and pathological conditions.And explore the role and potential mechanism of circ RNAs in PH.It is hoped that our study can provide a meaningful biomarker for the prognosis of patients with PH.Furthermore,the role of circ RNAs in pulmonary vascular remodeling,can be further elucidated,providing new ideas for the diagnosis,treatment and prognosis of PH.Methods1.The PBMCs of six patients with IPAH and six healthy controls were used as discovery cohorts and whole transcriptome sequencing was performed to obtain differentially expressed circ RNAs.The differentially expressed circ RNAs were verified by real-time PCR(q RT-PCR)in a large sample of PBMCs from IPAH patients and healthy controls,so as to search for circ RNAs with significance for IPAH.Clinical data of patients and healthy controls were collected for further statistical analysis to explore the value of newly discovered meaningful circ RNAs in prognosis of patients.IPAH patients and Chronic obstructive pulmonary disease associated pulmonary hypertension(COPD-PH)were also collected.Lung tissues and plasma samples from patients with IPAH and COPD-PH and healthy controls were further used to verify PBMCs results in IPAH patients,so as to find more meaningful circ RNA as biomarkers.2.The reverse splicing sites of circ RNAs was determined by Sanger sequencing;The quantification and localization of circ RNAs in tissues and cells were determined by absolute quantitative PCR,q RT-PCR and fluorescence in situ hybridization.Circ RNAs overexpression plasmid and si RNA were constructed,and the effects of circ RNAs overexpression and silencing on PAECs proliferation,migration and apoptosis were explored by CCK8 Kit,Brd U Kit,cell scratches,Transwell assay,AMPI assay and TUNEL assay.3.Combined with bioinformation analysis and RNA-Seq results,the downstream mi RNAs and target genes of circ RNAs were screened,and the binding between circ RNAs and mi RNAs as well as the binding between mi RNAs and m RNA were verified by dual luciferase reporting assay.Mimics and inhibitors of mi RNAs and its target gene m RNA were designed,and corresponding mimics or inhibitors were transfected.The effects of overexpression and knockdown of mi RNAs and its target gene m RNA on the proliferation,migration and apoptosis of PAECs were investigated by CCK8 Kit,Brd U Kit,cell scratches,Transwell assay,AM-PI assay and TUNEL assay.To reveal the influence of circ RNAs on PAECs function and related possible mechanisms.4.Monocrotaline(MCT)-PH rat model was constructed,tracheotomy was given to circ RNAs adeno-associated virus,cardiac function was evaluated by color doppler echocardiography,and hemodynamic indexes were evaluated by right cardiac catheterization.The effects of circ RNAs on MCT-PH in rats were investigated by q RTPCR,HE staining,Masson staining and α-SMA immunohistochemistry.Results1.The top 10 circ RNAs with the most obvious difference in whole transcriptome sequencing of PBMCs in IPAH patients were selected.The large sample verification results showed that circ GSAP was down-regulated in PBMCs in IPAH patients.Moreover,the expression level of circ GSAP in lung tissues and plasma samples of IPAH patients and COPD-PH patients was also decreased.Combined with clinical data analysis of patients,circ GSAP in PBMCs was found to be a potential biomarker for predicting the death of IPAH patients.Survival analysis showed that the lower the expression of circ GSAP in PBMCs,the worse the prognosis of IPAH patients.Meanwhile,circ GSAP in plasma is a potential biomarker for predicting death in patients with IPAH and COPD-PH.The lower the circ GSAP in plasma,the worse the prognosis of IPAH and COPD-PH patients.2.Circ GSAP is located on human chromosome 7,Chr7:77349351＿77355647＿-,which is composed of exon 15,exon 16,exon 17,exon 18 and exon 19 of parent gene GSAP(NM＿001350897.2).The total length is 518 bp.Quantitative and localization analysis of circ GSAP showed that it was mainly expressed in the cytoplasm of PAECs.Whether PAECs are cultured under normoxic or hypoxic conditions,overexpression of circ GSAP can inhibit the proliferation,migration and apoptosis resistance of PAECs,while silencing of circ GSAP expression can promote the proliferation,migration and apoptosis resistance of PAECs.3.In terms of mechanism,the dual luciferase report verified the binding of circ GSAP with mi R-27a-3p,and BMPR2,as the downstream target gene of mi R-27a-3p,was regulated by circ GSAP and mi R-27a-3p.The expression level of mi R-27a-3p was up-regulated in the lung tissues in IPAH patients and hypoxic-treated PAECs,and down-regulated in the PAECs of overexpressed circ GSAP.Transfection with mi R-27a-3p inhibitors inhibited PAECs proliferation,migration,and apoptotic resistance,and alleviate the effect of circ GSAP knockdown on the function of PAECs.The expression of BMPR2 m RNA was down-regulated in hypoxic-treated PAECs and IPAH patients,and up-regulated in overexpressed circ GSAP PAECs.Knocking down the expression of BMPR2 could promote the proliferation,migration and apoptosis resistance of PAECs,and alleviate the effect of mi R-27a-3p inhibitors on the function of PAECs.4.In vivo experiments,circ GSAP adeno-associated virus was applied to MCT-PH rats,and it was found that overexpression of circ GSAP could alleviate disease progression and right ventricular remodeling in MCT-PH rats,reduce the expression of mi R-27a-3p,and up-regulate the expression of BMPR2,SMAD1,SMAD5,SMAD8 and IOD3 m RNA.Conclusions1.Circ GSAP expression was down-regulated in PBMCs of IPAH patients,and down-regulated in lung tissues and plasma of IPAH patients and COPD-PH patients.Moreover,lower levels of circ GSAP are associated with poor prognosis of IPAH and COPD-PH,suggesting that circ GSAP may be an emerging biomarker for the diagnosis and prognosis of IPAH and COPD-PH patients.2.Circ GSAP is formed by reverse splicing of the parent gene GSAP exon 15-exon19,and is mainly expressed in the cytoplasm of PAECs,while the expression of circ GSAP is decreased in PAECs treated with hypoxia.Overexpression of circ GSAP can inhibit the proliferation,migration and apoptosis resistance of PAECs.Knockdown of circ GSAP could promote the proliferation,migration and apoptosis resistance of PAECs.3.Mi R-27a-3p inhibitors can inhibit the proliferation,migration and apoptotic resistance of PAECs,which can be reversed by silencing circ GSAP.Knockdown of BMPR2 expression promotes proliferation,migration,and apoptotic resistance of PAECs,which can be reversed by mi R-27a-3p inhibitors.Circ GSAP can increase BMPR2 m RNA expression in PAECs by competitively binding mi R-27a-3p,thereby reducing pulmonary artery intima injury,which may provide potential for PH treatment strategies.4.Overexpression of circ GSAP in vivo can alleviate the progression of disease and pulmonary vascular remodeling in MCT-PH rats.Meanwhile,the expression of mi R-27a-3p was decreased and the expression of BMPR2 signaling pathway was upregulated,which further demonstrated the potential therapeutic value of circ GSAP in PH pulmonary vascular remodeling by regulating mi R-27a-3p/BMPR2 signaling pathway.