The Functional Expression And Regulatory Network Of MiR-508-3p In Pulmonary Arterial Hypertension

1.IntroductionPulmonary arterial hypertension(PAH)featured serious cardiopulmonary-related chronic disease.It is presented by cell over-proliferation and fibrosis of pulmonary arterioles and muscularization of non-muscular arterioles,leading to pulmonary vascular resistance(pulmonary vascular resistance,PVR)gradually increased.A clear diagnosis of PAH requires measurement of average pulmonary artery pressure ≥25 mmHg,pulmonary wedge pressure≤15 mmHg and PVR>3 Wood in the right heart floating catheter test under sea level conditions.In addition,other causes of pulmonary hypertension must be excluded,such as lung disease and chronic thromboembolic disease.In the past 2 decades,the epidemiological and treatment concepts of PAH have undergone great changes.PAH is increasingly diagnosed in elderly patients,most of which have comorbidities,which increases the challenge of medical care.Although the pathogenesis of PAH begins with the pulmonary circulation,right heart failure is the leading cause of morbidity and death.Currently,the main clinical treatment strategy for PAH is to target the dysfunctional signaling pathways and conduction pathways in the pulmonary vascular system.The curative effect is limited,and the quality of life of PAH patients cannot be improved satisfactorily.Therefore,there is an urgent need to further explore the complex regulatory network of PAH and insight into novel specific therapeutic targets.PAH is characterized by a continuous increase in pulmonary artery pressure,which eventually leads to right heart failure and death.The mainly pathological features of PAH unveiled pulmonary artery vasoconstriction,remodeling and thrombosis.The molecular mechanism of PAH involves endothelial cell dysfunction,pulmonary artery smooth muscle cell proliferation,vascular inflammation and immune disorders,energy metabolism and mitochondrial abnormalities,excessive growth factor stimulation and ion channel defects.The imbalance of nitric oxide,endothelin-1,prostacyclin and serotonin and other vasoactive mediators in the body is common promotes pulmonary vasoconstriction.Vascular damage and endothelial cell dysfunction can change blood vessels by changing cytokine/growth factor levels,improving blood clotting ability,and increasing secretion of extracellular matrix(mainly elastin and collagen)homeostasis,which in turn promotes pulmonary vascular remodeling.The inflammatory response runs through the pathophysiological process of PAH and is considered to be the main pathogenic factor of pulmonary vascular remodeling.Pulmonary vascular cells,such as smooth muscle cells and endothelial cells,along with inflammatory cells are both viewed as essential pool of local chemokines and cytokines,which can pave the way for pulmonary vascular remodeling.In fact,increased expression of cytokines and chemokines leads to excessive VSMCs’ contraction and proliferation.IL-1 triggered the expression of fibroblast growth factor-2,FGF-2 and IL-6 can stimulate the proliferation of smooth muscle-like cells and pulmonary vascular fibroblasts in PAH.A variety of cytokines can directly control the cell proliferation,migration and differentiation of pulmonary vascular cells.Among them,IL-6 serves as a dominant player in these pro-inflammatory factors,which related to the pathogenesis of PAH.In animal experiments,compared with control group,recombinant IL-6 significantly enhanced pulmonary vascular remodeling in model mice and rised the hyperresponsiveness of model mice to hypoxia.In addition,IL-6 promotes the expression of FGF-2 by regulating the transcription factor KLF-5,which in turn leads to the proliferation of pulmonary artery smooth muscle cells.Perivascular cells composed of macrophages,T cells and B cells are infiltrated around the pulmonary artery vascular plexus in PAH.In the monocrotaline-induced PAH model,matrix-derived factor-1 is one of the key chemokines for inflammatory cell recruitment,and has the effect of blocking the pulmonary vascular infiltration of CD68+cells,CD3+cells and mast cells.The aggregation and activation of macrophages and monocytes usually leads to the release of cytokines,vasoactive molecules(such as endothelin)and reactive oxygen species(ROS).Activated macrophages secrete matrix metalloproteinases(MMP)and serine proteases,degrade basement membrane and extracellular matrix,and promote leukocyte migration,outer membrane fibrosis and cell differentiation.Activated macrophages can also enhance the ability of T cells to present antigens.The infiltration of CD8+T cells around pulmonary blood vessels is related to the impaired endothelial cell apoptosis and anti-apoptotic signal transduction.Bone morphogenetic protein(BMP)activates the expression of macrophages,and the macrophages themselves feedback and participate in BMP signal regulation.In PAH,BMPR2 mutations and BMPR2-related signaling pathway dysfunction can lead to inappropriate expression of growth factors and pro-inflammatory responses in vascular cells.MicroRNA(miRNA)is a non-coding RNA stretched a length of roughly 22 nucleotides.Several published researches indicated that miRNA engages in and regulates series of cellular bio-processes in normal cells and tumor cells,and abnormally expressed miRNAs regulate the pathological mechanism of diseases by acting as disease inhibitors or activators.So far,a variety of miRNAs,such as miR-17-92,miR-143/145,have been confirmed to play a role in regulating PAH-related signal pathways and pathological development.The important regulatory role makes miRs gradually become a new idea for clinical prediction and treatment of PAH.Our previous analysis found that miRNA expression chips sequenced in PAH tissues showed a significantly lower expression of miR-508-3p in the PAH group compared with the control group.This suggests that miR-508-3p may be involved in regulating and inhibiting PAH-related signal pathways and disease development.By studying the mRNA microarray of PAH pathological tissue sequencing,we found that in the PAH group,the expression of NR4A3(nuclear receptor subfamily4 A3)was hugely higher than that in the control group,indicating that the overexpression of NR4A3 may fuel the progression of PAH.Previous works suggested that miR-508-3p inhibits the proliferation and migration of cancer cells in ovarian cancer and renal clear cell carcinoma,and serves as an anti-cancer biomarker.Bioinformatics studies have found that miR-508-3p and the 3’-UIR of NR4A3 have a direct binding pair of base sequences,which suggests that miR-508-3p may directly regulate NR4A3’s function.However,the research on miR-508-3p in PAH is very limited.Can miR-508-3p affect the proliferation and migration of PASMC and act on NR4A3,thereby regulating PASMC function and influence on the development of PAH still need to be discussed.In summary,this subject proposes the following hypothesis:specific overexpression of miR-508-3p in.PASMC,knock-down NR4A3 expressed give rise to a decrease in the proliferation and migration ability of PASMC,thereby inhibiting the occurrence of PAH.This study will verify the expression changes of NR4A3 by constructing a monocrotaline-induced PAH rat model.At the same time,miR-508-3p mimics/inhibitor was used to simulate miR-508-3p overexpression and knockdown on human-derive PASMC(h-PASMC)to explore miR-508-3p-NR4A3 regulation mechanism of the pathway provides in vivo and in vitro experimental evidence support for finding new molecules for the treatment of PAH.2.Objectives(1)To study the role and mechanism of miR-508-3p expression and regulation in h-PASMC;(2)To study the effect and mechanism of miR-508-3p-NR4A3 in MCT-PAH rats and h-PASMC.3.Methods3.1 PAH tissue sample gene expression microarray and miRNA expression matrix analysisThe Gene Expression Omnibus(GEO)platform obtains two PAH-related pathological tissue gene sequencing expression profiles and two miRNA expression matrices,and obtains differential genes and differences through bioinformatics methods miRNA.Construct a miRNA-mRNA relationship and interaction network to screen out key miRNAs and target genes.3.2 Construction of MCT-induced pulmonary arterial hypertension rat modelSprague Dawley(SD)rats(average weight 180±200g)were divided into two groups,each with 5 rats.Each rat in the experimental group was injected with monocrotaline(60 mg/kg;C2401,Sigma-Aldrich)subcutaneously in the left lower abdomen once a day for 3 weeks.In the control groups,rats were injected with equivalence of normal saline in the left lower abdomen once a day for 3 weeks.Confirm that the modeling is successful:3 weeks later,the rats are anesthetized and connected to a ventilator,and the mean pulmonary arterial pressure(mPAP),right ventricular systolic pressure(RVSP)and right ventricular hypertrophy index(Right ventricular hypertrophy index,RVHI).3.3 Cell culture(1)Determination of miR-508-3p functional expression:after platelet-derived growth factor-BB(PDGF-BB,GF149,Sigma-Aldrich,USA)stimulated cells(not added in the control group),at 0,6,12 and 24h,respectively.The total cell RNA was extracted,and the expression of miR-508-3p was calculated by real-time fluorescent polymerase chain reaction(qRT-PCR).(2)miR-508-3p transfection:the overexpression group and the inhibition group were transfected with miR-508-3p mimics and inhibitor,respectively.The control group was not treated,and the same amount of transfection reagent was added to the carrier group.(3)CCK-8 experiment:Cells were transfected with miR-508-3p mimics and inhibitor respectively.The control group was left without any treatment.Cell viability was measured at 1,2,4,8,12,24 and 48h.(4)Wound healing experiment:The cells were transfected with miR-508-3p mimics and inhibitor,and the control group was left untreated.Observe and measure the area of the scar at 0 and 48h.(5)Immunofluorescence experiment:The cells were transfected with miR-508-3p mimics and inhibitor respectively,and the control group was left untreated.Calculate the nuclear ratio of ki67.(6)Double luciferase gene report experiment:It is proved that NR4A3 is the downstream direct acting target gene of miR-508-3p.3.4 Western blotTo detects the expression of NR4A3 and GAPDH at the protein level.3.5 qRT-PCRTo detects the expression of miR-508-3p,miR-508-3p mimics,miR-508-3p inhibitor,NR4A3,GAPDH at the mRNA level.4.Results4.1 Bioinformatics methods screen out key miRNAs and their target genes involved in and regulating PAHWe screened two PAH-related gene expression chips(GSE70456,GSE117261)and two miRNA expression matrices(GSE55427 and GSE67597)from GEO.After analysis and screening,we obtained 185 differentially expressed genes(dysregulated expression genes,DEG)and 172 differentially expressed miRNAs(dysregulated expression miRNA,DEM),through protein-protein interaction(PPI)and miRNA-mRNA regulatory network,we finally decided to explore the role of miR508-3p and its downstream target gene NR4A3 in PAH disease development and signal regulation.4.2 Successfully established the MCT-PAH rat modelFive rats in the PAH group were injected with MCT for 3 consecutive weeks.Five rats in the control group were injected with saline for 3 weeks.According to the measurement results of the determination indicators,the values of mPAP,RVSP and RVHI in the PAH group were significantly higher than those in the control group,and were statistically significant.It shows that the PAH rat model induced by MCT is successfully established.4.3 Cell culture(1)After PDGF-BB stimulated h-PASMC,we found that the expression of miR-508-3p decreased gradually at 0,6,12 and 24h.(2)Under the condition of miR-508-3p mimics transfection,transfection of miR-508-3p inhibitor significantly inhibited the expression of miR-508-3p(p<0.05).(3)In the CCK-8 experiment,compared with the control group,transfection of miR-508-3p mimics inhibited the proliferation of h-PASMC,and transfection of miR-508-3p inhibitor increased the proliferation of h-PASMC(p<0.05).The scratch suggested that the miR-508-3p inhibitor transfected cells had stronger migration ability(p<0.05).(4)ki67 is a generally accepted marker of cell proliferation.We found that h-PASMC transfected miR-508-3p mimics reduced the fluorescence intensity and nuclear ratio of ki67,while the fluorescence intensity of ki67 and nuclear ratio increased after the cells were treated with miR-508-3p inhibitor compared with the control group.(P<0.05).(5)We found that in the pulmonary artery vascular tissue of rats in the PAH group,NR4A3 showed high expression at both protein and mRNA levels.The dual luciferase gene report experiment showed that when the cells were transfected with miR-508-3p mimics,the fluorescence intensity when combined with wild-type NR4A3 was significantly weaker than when combined with mutant NR4A3.At Western blot and mRNA levels,we found that miR-508-3p mimics transfected cells inhibited the expression of NR4A3,and cells transfected with miR-508-3p inhibitor promoted the expression of NR4A3.These all proved that miR-508-3p directly acts on NR4A3.(P<0.05).5.Conclusion(1)The expression of miR-508-3p is low in PAH,while the expression of NR4A3 is high,which is consistent with the results of our bioinformatics research;(2)NR4A3 is a direct downstream target of miR-508-3p,miR-508-3p-NR4A3 may play a role in the disease development and signal regulation pathway of human PAH;(3)Overexpression of miR-508-3p inhibits the proliferation and migration of h-PASMC,directly regulates the expression of NR4A3 and activates the MEK pathway to play biological functions in PAH,which can be used as a new idea for clinical treatment of PAH.