The Effect And Mechanism Of Circ-grm1 In The Proliferation And Migration Of Hypoxic Pulmonary Artery Smooth Muscle Cell

BackgroundPulmonary arterial hypertension(PAH,or PH)is a group of "cancer like" symptom characterized by increased pulmonary vascular resistance and pulmonary vascular remodeling caused by different reasons.If it is not treated in time,the clinical prognosis is very poor.The causes of this disease are complex and unclear today,which may be related to congenital abnormality,genetic susceptibility,hypoxia and metabolic abnormalities.The pathogenesis of pulmonary arterial hypertension is complicated.Pulmonary small vessel contraction,thrombosis development and pulmonary vascular remodeling are involved in the pathogenesis of PAH.Pulmonary vasoconstriction is an important factor in the pathogenesis of PAH.Vasoconstriction is mainly caused by the imbalance of vasoactive factors.The normal contraction of blood vessels is maintained by three pathways:prostacyclin,endothelin-1 and nitric oxide[1].In PAH,these three pathways are imbalanced.Microthrombosis plays an important role in the progression of pulmonary arterial hypertension.Pulmonary vascular remodeling is a key factor in the pathogenesis of PAH.Pulmonary vascular remodeling involves uncontrolled proliferation,altered metabolic,clonal expansion,and resistance to programmed death.At present,the treatment of PAH mainly focuses on anti-vasoconstriction and has limited effect on improving pulmonary vascular reconstruction.Drug therapy mainly includes calcium channel receptor blockers,endothelin receptor antagonists(ERA),prostaglandin analogues and prostacyclin receptor agonists,soluble guanylate cyclase(SGC)and phosphodiesterase-5 inhibitors.The clinical prognosis of most patients has improved greatly,but some patients still manifest poor effect on multiple drugs.There is no definite single targeted treatment to solve pulmonary vascular remodeling.Therefore,paying more attention to the potential molecular mechanism in the process of pulmonary arteriolar remodeling may bring groundbreaking progress to the treatment of pulmonary arterial hypertension.With the continuous development of mcroarray and next-generation sequencing technology,many functions of a variety of non protein coding RNAs in diseases have attracted much attention.Among them,microRNA,long noncoding RNA and circular RNAs(circRNAs)became new regulators of vascular function,especially in hypertension and PAH.CircRNAs are novel RNA molecules rings formed by lassodriven or intron-paired driving circularization.Because circRNAs lack terminal 5' end cap and 3' polyadenylation tail,circRNAs are very stable and difficult to be separated by ribonuclease R(RNase R)and other exonucleases[2-4].A large number of extensive and abundant circRNAs were found[5-8].Many biological functions and characteristics of circRNAs have also been gradually recognized.CircRNAs are involved in many disease processes,such as vascular dysfunction,vascular development,vascular growth and remodeling.CircRNAs imbalance is involved in vascular reconstruction through a variety of mechanisms,such as phenotypic transformation,proliferation and migration.It is suggested that circRNAs may be involved in the occurrence and progression of PAH by regulating vascular remodeling.As a new member of noncoding RNA,circRNAs may be a new target for PAH diagnosis and treatment.In this study,pulmonary artery smooth muscle cells(PASMCs)were used to simulate the cell model of PAH.According to the results of RNA sequencing,the differentially expressed circRNAs and mRNAs were screened.We found the expression of mmu_circ₀001907 in hypoxic PASMCs was significantly up-regulated.Its parent gene was glutamate receptor metabotropic 1(Grm1),which was a potential molecule to regulate the function of pulmonary artery smooth muscle cells.CircRNA-Grml(mmu_circ₀001907)was selected as a representative to study the role of circRNAs in the proliferation and migration of hypoxic PASMCs,and preliminarily explore the molecular mechanism of Grml targeted regulation by binding with RNA binding protein Fus,so as to provide a new molecular target for the study of hypoxic PAH.Part ?:Screening and validation of differentially expressed circRNAs in hypoxic pulmonary smooth muscle cellsObjective:The hypoxic PASMCs model was established.High-throughput sequencing was used to detect the differentially expressed circRNAs,and the target circRNA was selected.To investigate the effects of target circRNA on proliferation and migration of hypoxic PASMCs.Methods:1.Primary PASMCs were cultured and identified.2.PASMCs were treated with hypoxia(3%O2)to establish the cell model of hypoxic PASMCs.Total RNA was extracted.CircRNAs cultured in normoxia and hypoxia were detected by high-throughput sequencing,and circRNAs with significantly different expression were screened.3.The circ-Grml specific siRNA was transfected into PASMCs to knock down the expression of circ-Grml,and the effect of circ-Grml on proliferation and migration of PASMCs was explored.3.1 Detecting the transfection efficiency of siRNAs through quantitative Real-time polymerase chain reaction(qRT-PCR).3.2 The effect of circ-Grml on proliferation of PASMCs was detected by CCK-8 and EdU test.3.3 The effect of circ-Grml on migration of PASMCs was detected by scratch and transwell test.3.4 Flow cytometry was used to detect the effect of circ-Grml on PASMCs cell cycle.3.5 The effects of circ-Grml on cell migration related proteins matrix metalloproteinase(MMP)2,MMP9 and proliferation related protein cyclin A,proliferating cell nuclear antigen(PCNA),cyclin dependent kinase 1(CDK1),Ki67 protein expression were detected by western blot(WB).Results:1.Immunofluorescence test confirmed that the cultured cells were smooth muscle cells.The expression rate of ?-SMA was higher than 97%in cultured cells,indicating that the primary PASMCs had high purity.2.The high-throughput sequencing results showed that taking the normal cultured pulmonary artery smooth muscle cells as control,13 circRNAs with up-regulated expression and 9 circRNAs with down-regulated expression were screened in hypoxia pulmonary artery smooth muscle cells.The cluster heat map of differentially expressed genes was made.Mmu-circ-0001907(Circ-Grm1)was selected as the target circRNA.3.The effect of cicr-Grml on proliferation and migration of PASMCs.3.1 Quantitative real-time polymerase chain reaction(QRT-PCR)confirmed that after transfection of si-circ-Grm1,the relative expression of circ-Grml decreased significantly compared with si-NC group(**P<0.01),suggesting that the transfection was effective.3.2 Taken the normoxia group as control,CCK-8 and EdU both showed that the proliferation of PASMCs in hypoxia group(si-NC,si-circ-1,si-circ-2)increased significantly(**P<0.01).After knockdown of circ-Grml,cell proliferation was significantly weaker than those in si-NC group(**P<0.01).3.3 Cell scratch test and transwell test showed that compared with normoxia group,the cell migration ability of PASMCs in hypoxia group(si-NC,si-circ-1,si-circ-2)was significantly enhanced(**P<0.01).After knockdown of circ-Grml,the cell migration ability decreased significantly compared with si-NC group(**P<0.01).3.4 Flow cytometry confirmed that compared with normoxia group,the process of cell cycle of hypoxia group(si-NC,si-circ-1,si-circ-2)was accelerated and cell proliferation was increased significantly(**P<0.05).Compared with si-NC group,cell cycle progression was blocked and cell proliferation was inhibited(**P<0.05).3.5 Compared with normoxia group,the expressions of cyclin A,PCNA,CDK-1,Ki67,MMP2 and MMP9 in hypoxic(si-NC,si-circ-1,si-circ-2)group were significantly increased(**P<0.01).Compared with si-NC group,the expression of cyclin A,PCNA,CDK-1,Ki67,MMP2 and MMP9 in PASMCs decreased significantly after knockdown of circ-Grml(**P<0.01).Part II:Investigating the mechanism of circ-Grml in regulating hypoxic pulmonary artery smooth muscle functionObjectiveThe present study trying to predict and verify the RNA binding protein(RBP)of circGrm1.And furthermore exploring the effect of circ-Grml binding with RBP on the stability and expression of target gene Grm.Besides,preliminarily studying the molecular mechanism of circ-Grm1 regulating the function of pulmonary artery smooth muscle cells.Method1.High throughput sequencing was used to detect the differentially expressed mRNAs in PASMCs of hypoxia group and normoxia group.2.Predict and confirm the RNA binding protein(RBP)of circ-Grm1.2.1 The RNA binding protein(RBP)of circ-Grml was predicted by Starbase,and the binding abundance between circ-Grml and RBP was predicted by RNA protein interaction prediction(RPISeq).2.2 The binding of circ-Grml and Fus was verified by RNA immunoprecipitation(RIP)and pull-down experiment.3.The effect of circ-Grml combined with Fus on the stability and expression of Grml mRNA was explored.3.1 Actinomycin D was used to detect the effect of the combination of circ-Grml and Fus on the stability of Grml mRNA.3.1.1 Down-regulate the expression of FUS and circ-Grml through small interfering RNA.Then,the transfection efficiency of siRNAs was tested.3.1.2 Actinomycin D test was used to investigate the effect of the combination of circ-Grml and Fus on the stability of Grml mRNA.The expression levels of Grml in different groups of cells were detected at different time points,and the degradation curve of Grm1 was drawn.3.2 WB was adopted to detect the effect of the combination of circ-Grml and Fus on Grml mRNA expression.The expression of Grml in different groups was examined by WB.4.The role of Grml in the regulation of circ-Grml on proliferation and migration of PASMCs was inspected.4.1 Up-regulating the expression of Grml and circ-Grml through plasmid transfection.The plasmid transfection efficiency was verified by qRT-PCR.4.2 WB was used to detect the effect of overexpression of circ-Grml and Grml on Grml expression.4.3 The effect of Grml on proliferation and migration of circ-Grml in hypoxic PASMCs were detected by CCK-8 test,EdU test,transwell test,western blot test and scratch test.5.The related signal pathways of circ-Grml regulating Grml expression were analyzed and verified.5.1 KEGG enrichment analysis was adopted to explore related signal pathways.5.2 WB was used to detect the related signal pathway protein expressions.Results1.RNA sequencing showed that 1644 mRNAs were up-regulated and 2098 mRNAs were down-regulated in hypoxic PASMCs compared with normoxic PASMCs.The target gene Grml of circ-Grml was retrieved from circbase.At the same time,the microarray results showed that the expression of Grml was down regulated in the differentially expressed target genes,which was consistent with the prediction of shengxin website,and Grm1 was determined as the target gene of circ-Grm1.2.Predicted and confirmed the RBP of circ-Grml was Fus.2.1 The Starbase website predicted the RBP of circ-Grml was Fus,and the random forests(RF)and support vector machine(SVM)scores of circ-Grml and Fus predicted by RPISeq were 0.51 and 0.65 respectively.The RF and SVM scores of Grml combined with Fus were 0.75 and 0.97 respectively.These results suggesting that circGrml and Grml might combine with Fus.2.2 Rip experiment and pull-down experiment confirmed that circ-Grm1 was highly combined with Fus.3.The combination of circ-Grml and Fus could inhibit the stability and expression of Grm1 mRNA.3.1 The combination of circ-Grml and Fus could inhibit the stability of Grm1 mRNA.3.1.1 QRT-PCR confirmed that the expression of FUS decreased significantly after transfection of si-Fus(**P<0.01);After transfection of Fus overexpression plasmid,the expression of Fus increased significantly(**P<0.01),which confirmed that the transfection was effective.3.1.2 Compared with the control group,the degradation of Grml mRNA in PASMCs decreased after circ-Grml knockdown(**P<0.01);The degradation of Grml mRNA in PASMCs increased significantly after Fus knockdown(**P<0.01).The present study confirmed that the combination of circ-Grml and Fus could inhibit the stability of Grm1 mRNA.3.2 Circ-Grml combined with Fus could inhibit Grm1 mRNA expression.After overexpression of Fus,the expression of Grml in PASMCs increased significantly(**P<0.01).It was suggested that the expression of Fus positively correlated with the expression of Grm1.Knockdown of circ-Grml significantly increased the expression of Grml in PASMCs(**P<0.01).The expression of Grml in si-circ-Grml and Si-Fus co transfection group was significantly lower than those in si-circ-Grm1 group(**P<0.01).The above results suggest that the combination of circGrml and Fus could inhibit the expression of Grml mRNA.4.Grm1 could reverse the promoting effect of circ-Grm1 on proliferation and migration of PASMCs.4.1 QRT-PCR confirmed that take the normal group as control,the expression of Grml was significantly up-regulated in Grml overexpression(p-Grml)group(**P<0.05).Besides,the expression of circ-Grm1 was significantly up-regulated in circGrml overexpression(oe-circ-Grm1)group(**P<0.05);The transfection was confirmed to be effective.4.2 WB showed that compared with the control group,the expression of Grml in circ-Grm1 overexpression group decreased significantly(**P<0.05);Compared with the circ-Grml overexpression group,the expression of Grml in circ-Grml and Grml overexpression group increased significantly(**P<0.05).4.3 CCK-8 and EdU analysis showed that compared with the overexpression group of circ-Grml,the cell proliferation of the co-overexpression group of circ-Grml and Grml was inhibited(**P<0.01).Cell scratch and transwell experiment showed that the migration ability of PASMCs increased significantly after overexpression of circ-Grml(**P<0.01),and decreased significantly after overexpression of circ-Grml and Grml(**P<0.01).WB showed that cell migration related proteins MMP2 and MMP9,proliferation related proteins cyclin A,PCNA,CDK-1 and Ki67 increased significantly after overexpression of circ-Grm1(**P<0.01),but decreased significantly after overexpression of circ-Grml and Grm1(**P<0.01).It is suggested that Grm1 could partially reverse the promoting effect of circ-Grml on proliferation and migration of hypoxic pulmonary artery smooth muscle cells.5.Circ-Grml regulated the Grml expression through the rhoptry associated protein1(Rap1)/extracellular signal-regulated kinase 1(ERK1)signal pathway.5.1 KEGG analysis showed that Rap1 signal pathway had the highest enrichment.5.2 The western blotting results indicated that the protein expression levels of Grml and Rap1b in the hypoxia groups were lower compared with those of the normoxia group,whereas these were increased in cells overexpressing Grml compared with oecirc-Grm1 group(**P<0.01).Conversely,the expression level of ph-ERK1 in the hypoxia groups was higher compared with the control group,whereas it was lower in cells with overexpressing Grml compared with oe-circ-Grml group(**P<0.01).In addition,the co-transfection of oe-circ-Grml and p-Grml reversed the effects of Grml overexpression on the expression levels of Grml,Rap1b and ph-ERK1.Conclusion:1.Circ-Grml was highly expressed in hypoxic PASMCs;Circ-Grml could promote the proliferation and migration of hypoxic PASMCs.2.The combination of circ-Grm1 and RNA binding protein Fus inhibited the stability and expression of Grml mRNA.3.Grml could reverse the promoting effect of circ-Grml on the proliferation and migration of hypoxic PASMCs.4.Circ-Grml combined with Fus could inhibit the expression of Grml by regulating Rapl/ERK1 signal pathway and promote the regulation of hypoxic PASMCs proliferation and migration.