Mechanism Of MiR-203a-3p Regulating The Proliferation And Migration Of PASMCs Through PIK3CA/PI3K/Akt Pathway In PAH

Background:Pulmonary arterial hypertension（PAH）is a malignant pulmonary vascular disease characterized by elevated pulmonary vascular resistance and pulmonary artery pressure,which eventually leads to right ventricular dysfunction,right heart failure and even death.The pathogenesis of PAH is still uncertain.Pulmonary artery remodeling is an important pathological feature of PAH,mainly manifested as excessive proliferation,migration,and aggregation of pulmonary artery smooth muscle cells（PASMCs）.This tumor-like appearance is associated with changes in gene expression.Micro RNA（miRNA）are highly conserved non-coding RNAs that can negatively regulate the expression of target genes,and participate in the occurrence and development of PAH via various signaling pathways.miR-203a-3p regulates various biological processes such as cell proliferation and migration,and participates in tumor growth,angiogenesis,and cardiac fibrosis.The target genes and their downstream signaling pathways of miR-203a-3p are also involved in the pathophysiology of PAH.At present,there are few studies on miR-203a-3p in PAH,and the role and mechanism of miR-203a-3p in PAH are still unclear.Objective:This project aims to study the expression and role of miR-203a-3p in idiopathic PAH（Idiopathic PAH,IPAH）patients and PAH rat models,and explore the molecular mechanism of miR-203a-3p.Methods:1.Detection of miR-203a-3p expression in plasma from IPAH patientsPatients with IPAH who were first diagnosed in the Department of Cardiovascular Medicine of the Second Xiangya Hospital from September 2019 to September 2021 were continuously collected,and healthy volunteers served as the control group.Plasma miRNA was extracted,the expression level of miR-203a-3p in plasma was detected by RT-qPCR,and the correlation between miR-203a-3p and clinical data was analyzed.2.Detection of miR-203a-3p expression in PAH rat model180-220g male Sprague-Dawley（SD）rats were selected to construct the PAH rat model of monocrotaline（MCT）and Su5416 combined with chronic hypoxia（10%O₂,Su/Hx）,and the right heart catheterization was used to identify the model.（1）MCT-induced PAH（MCT-PAH）rat model:according to the experimental design,all experimental animals were randomly divided into control group,three-week（3w）group,and four-week（4w）group.The control group and the experimental group were injected with normal saline or MCT（60mg/Kg）,PAHs were identified at the 3rd/4th week of modeling,and whole blood,lung tissue,and heart were harvested to detect the expression of miRNA-203a-3p in plasma and lung tissue at different time points.（2）Su/Hx-induced PAH rat model:according to the experimental design,all experimental animals were randomly divided into normoxia group,3w group,and eight-week（8w）group,and the normoxia group and the experimental group were injected with solvent or Su5416（20mg/Kg）,normoxic and Su/Hx rats were placed in normoxic and hypoxic environments,respectively.The 3w group and the 8w group were identified at the end of the 3rd and 8th weeks,and the whole blood,lung tissue and heart were harvested to detect the expression of miRNA-203a-3p in plasma and lung tissue at different time points.3.Detection of the expression and role of miR-203a-3p in PASMCsPrimary PASMCs were extracted from healthy rats.The effects of hypoxia（1%O₂）or PDGF-BB（30ng/ml）stimulation on the proliferation,migration and miRNA-203a-3p expression of PASMCs were evaluated.Using miR-203a-3p mimic to over express miR-203a-3p to evaluate the role of miR-203a-3p in the proliferation and migration of PASMCs.4.Evaluation of the preventive and therapeutic effects of miR-203a-3p on PAH rats（1）The miR-203a adeno-associated virus（miR-203a-AAV5）or negative control group（miR-203a-NC）was delivered through tracheal puncture,and the MCT-PAH or Su/Hx model was established 3 days later.MCT and Su/Hx rats were identified at modeling 4W and 8W,respectively,to evaluate the preventive effect of miR-203a-3p on PAH rats.（2）In the fourth week of Su/Hx rats,that is,after being transferred to normoxia for 1 week,miR-203a-AAV5 or miR-203a-NC were delivered through the trachea.Identification of models at week 9 to assess the therapeutic effect of miR-203a-3p on Su/Hx-PAH.5.Exploring the mechanism of miR-203a-3p in PAHThe lung tissue of Su/Hx rats treated with miR-203a-AAV and PASMCs treated with hypoxia were selected to detect the expression differences of potential targets of miRNA-203a-3p and its downstream molecular pathways.The dual-luciferase reporter assay was used to verify the target gene,and its effect on the proliferation and migration of PASMCs was evaluated by regulating the expression of the target gene.Results:1.A total of 51 patients with IPAH（37.61±12.65 years old,35women（68.6%））were included in this study.Compared with healthy controls,the expression of miRNA-203a-3p in the plasma of IPAH patients was significantly decreased（p<0.05）,and miRNA-203a-3p was positively correlated with TAPSE,S’,RV-FAC and CO（r=0.349,0.313,0.456,respectively,all P<0.05）;while they were negatively correlated with NT-Pro BNP,m PAP,m RVP,and PVR（r=-0.546,-0.509,-0.456,-0.622,respectively,all P<0.01）.2.The expression of miR-203a-3p in the plasma and lung of MCT-and Su/Hx-induced PAH rats were significantly reduced,and its expression in the plasma and lung of Su/Hx-induced PAH rats was decreased progressively over time.3.Hypoxia stimulated the proliferation of PASMCs,accompanied by the conversion of PASMCs from contractile type to synthetic type.Overexpression of miR-203a-3p by mimic inhibited the proliferation and phenotype conversion of PASMCs.PDGF-BB promoted the migration of PASMCs,which could be inhibited by overexpressed miR-203a-3p.4.In prevention and treatment schedule,over expression of miR-203a-3p through airway delivery of miR-203a-AAV significantly reduced right ventricular systolic pressure,pulmonary artery medial thickness and Fulton index in Su/Hx-treated rats（p<0.05）.In MCT-PAH rats,over expression of miR-203a-3p only slightly decreased RVSP and Fulton index,but the difference was not statistically significant（p>0.05）.5.RT-qPCR in lung and PASMCs showed that miR-203a-3p significantly down-regulated the expression of PIK3CA,PI3K and Akt.The dual-luciferase reporter confirmed that PIK3CA is a direct target gene of miR-203a-3p.Further analysis indicated that down-regulated PIK3CA inhibited the proliferation and migration of PASMCs by down-regulating the activation of PI3K/Akt.Conclusion:（1）The expression of miRNA-203a-3p was significantly down-regulated in IPAH patients and PAH rats.The level of miRNA-203a-3p in IPAH patients was negatively correlated with hemodynamic parameters and positively correlated with right ventricular function.miR-203a-3p may be an effective biomarker for IPAH patients.（2）miR-203a-3p inhibits the proliferation and migration of PASMCs by targeting PIK3CA/PI3K/Akt pathway,preventing and rescuing Su/Hx-induced PAH.Figures:21,Tables:10,References:113.