| 【Backgroud and Aims】Idiopathic Pulmonary hypertension（IPAH）is a kind of malignant cardiopulmonary vascular disease,charactered by pulmonary vascular remodeling and the continuous increase of pulmonary vascular resistance（PVR）,causing the insufficiency of right heart and lung and eventually leading to right heart failure.The etiology and pathogenesis of this disease are still not that clear.IPAH belongs to the Group 1 of pulmonary hypertension（PH）,according to the Chinese Guidelines of Pulmonary Hypertension（2021 edition）and the clinical classification of PH recommended by the 6th World Pulmonary Hypertension Congress.IPAH is a rare disease with an reported incidence of 0.9 to 2.6 cases per million population per year abroad.Epidemiological data show that Chinese patients with IPAH are with an average age of only 36 years old,and most patients are female.The study showed that the median survival time of untreated patients with IPAH was just2.8 years.In recent years,although drug treatment has improved clinical symptoms and prognosis of patients to a certain extent,the high cost of drugs has caused heavy economic burden to patients’families,and it is common to become poor and return to poverty due to illness.In addition,randomized double-blind controlled clinical studies have shown that the best existing targeted drugs have less than 6mm Hg effect on mean pulmonary artery pressure.Furthermore,these drugs mainly target pulmonary vascular overcontraction rather than pulmonary vascular remodeling,suggesting that it is essential to further study the etiology and pathogenesis of pulmonary vascular remodeling in pulmonary hypertension and to find more targeted targets.The most distinctive pathophysiological feature of IPAH is pulmonary vascular remodeling,which means intima thickening,midia hypertrophy,accumulation of extracellular matrix and infiltration of various immune cells in tunica adventitia vasorum under the impact of hemodynamic changes（pull force,shear stress）,mechanical damage and various factors in the circulation.Pulmonary artery smooth muscle cells（PASMC）are important participants in media hypertrophy and pulmonary vascular remodeling.Compared with PASMC from healthy people,PASMC from IPAH patients showed pro-proliferation,pro-migration,and anti-apoptotic phenotypes.Antagonizing these phenotypes through target drug or gene modification interventions can improve pulmonary vascular remodeling and pulmonary hypertension in PH animal models.Previous studies have also suggested that emerging role of non-coding RNA,including micro RNA（mi RNA）and long non-coding RNA（lnc RNA）,are closely correlated with the pathogenesis and prognosis of IPAH.Moreover,the expression and function of non-coding RNAs such as mi R29c and mi R376a are different in male and female IPAH patients,which can explain the"sex paradox"of IPAH,which is that IPAH patients are more female,but male patients have a poor prognosis.However,at present,the expression of circular RNA（circ RNA）,another non-coding RNA,in IPAH and its relationship with disease prognosis and pathogenesis are still being attractive.Circ RNA is a class of closed RNA with the lack of 3’and 5’structures,so that it is difficult to be digested and decomposed by RNA enzymes and is more stable than linear RNA.Current studies indicate that circ RNA has four main biological functions:I Adsorption of mi RNA to participate in post-transcriptional regulation of m RNA;II Interaction with RNA-binding proteins to modulate cell functions by affecting the expression of other proteins;III Translation of polypeptides involved in physiological and pathological processes of diseases;IV Regulation of the host genes.It is generally believed that when circ RNA is located in the cytoplasm,it is more likely to act as a mi RNA sponge or to play a role through translation peptides,while when circ RNA is located in the cytoplasm,it is more likely to interact with RNA-binding proteins or regulate the expression of the host genes.In recent years,a variety of circ RNAs have been found to be potential diagnostic and prognostic biomarkers of IPAH,most of which are involved into the pathology through the circ RNA/mi RNA/m RNA regulatory pathway,while few studies have focused on circ RNA participating in the occurrence and development of IPAH in other ways（e.g.,Interaction with RNA-binding proteins）.The whole-transcriptome sequencing of PBMC from collected from 6 IPAH patients and 6 healthy controls showed that 146 circ RNAs with significantly increased expression and 124 with significantly reduced level were displayed,of which circ METAP2 was highly expressed in PBMC of IPAH patients.Through literature review,we found that METAP2（Methionyl Aminopeptidase 2）,the host gene of circ METAP2,was expressed in pulmonary arterial of PH patients and PH animal models,and was highly expressed in PASMC,participating in the regulation of PASMC proliferation.However,no studies have explored the expression of circ METAP2 in IPAH,its correlation with the pathogenesis and prognosis of IPAH,its characteristics in IPAH patients of different genders,its functional impact on PASMC and its specific regulatory mechanism.In view of this,this study intends to determine the true expression of circ METAP2 in IPAH patients through real-time quantitative PCR,and to clarify the role of circ METAP2 in IPAH patients’phenotype,including hemodynamics and prognosis,as well as the potential sex difference,through a cohort study.Meanwhile,the effects of circ METAP2 on PASMC cell proliferation,migration and cell death were explored at the cellular level by constructing vector of circ METAP2overexpression.The sublocation and potential regulatory mechanism of circ METAP2in PASMC were further explored through RNA fluorescence in situ hybridization and bioinformatics analysis.In short,this study aims to clarify the expression and mechanism of circ METAP2 in IPAH,provide a new biomarker for the diagnosis and prognosis of IPAH,and provide new sight fo new potential targets treatment.【Methods】Accordingly,we conducted this study as three parts:Part IStudy of the expression of circ METAP2 in IPAH and the correlation with clinical indices and survival.First,the existence of circ METAP2 was verified by Sanger sequencing.Based on this,66 IPAH patients（28 males,38 females）and age-and-sex-matched 36 normal controls（16 males,20 females）were prospectively enrolled in the clinical study from May 2009 to April 2018 at our center,with Body mass index（BMI）matching between the male and female.The normal controls were all recruited at the general medical department at our center during the same time.Sex hormone levels of IPAH patients were measured.Including Estradiol（E2）,follicle-stimulating hormone（FSH）,Luteinizing hormone（LH）,Progesterone（P）and Testosterone（TT）.Samples were taken early in the morning after the 2nd day of hospitalization for the male,early follicle phase for the female at reproductive age（as days 1–7 from the first day of bleeding）,and 8-11 a.m,the next day for the female with amenorrhea.The expression of circ METAP2 in PBMC of IPAH patients and normal controls was determined by real-time quantitative PCR.Receiver Operating characteristic Curve（ROC Curve）was used to analyze the diagnostic value of circ METAP2 in the total cohort,male cohort and female cohort,and to determine the cut-off values of each group.The correlation between the relative expression of circ METAP2 and the hemodynamics of IPAH patients was analyzed by Pearson correlation analysis.The correlation between circ METAP2 relative expression and serum sex hormone level was analyzed by univariate/multiple linear regression analysis.The end of follow-up was malignant events（event,including,all-cause death,inceresed dosage of target drugs duo to the deterioration of cardiopulmonary function,changes of oral medication to intravenous medications or readmission,etc.）,event-free patients were followed up till December 20,2020.The Event-free survival rate was calculated by Kaplan-Meier method.Part IIThe expression of circ METAP2 in PASMC and lung tissue from MCT-induced PH rats and the impact on proliferation,migration and death of PASMC.The MCT-induced PH rats was established,and the expression of circ METAP2 in lung tissues was detected by real-time quantitative PCR.The plasmid of overexpressed circ METAP2 was constructed,and the negative control was the vehicle（Mock）.The constructed plasmid of overexpressed circ METAP2 was transfected into PASMC,and detect the overexpression efficiency after 48h culture.CCK-8 Kit was used to test proliferation of PASMC cultured under normoxic/hypoxic（21%O2/5%O2）cultures.The wound healing was used to detect migration of PASMC under normoxic/hypoxic（21%O2/5%O2）cultures.The Calcein-AM/PI live/dead cell staining kit was used to test cell death of PASMC under normoxic/hypoxic（21%O2/5%O2）cultures.Part IIIStudy on the mechanism of changed proliferation,migration and cell death of PASMC caused by circ METAP2.RNA fluorescence in situ hybridization（FISH）was used to detect the sublocalization of circ METAP2 in PASMC under normoxic/hypoxic（21%O2/5%O2）condition.The spatial characteristics of circ METAP2 and the protein SOX2 were studied by FISH combined with cell immunofluorescence.Western Blotting was used to detect the expression of SOX2 in PASMC under normoxic/hypoxic（21%O2/5%O2）conditions.The downstream genes of SOX2 were identified from the Gene Transcription Regulation Database（GTRD,http://gtrd.biouml.org）.GO clustering analysis and KEGG pathway analysis were used to analyze the aggregation of downstream genes.DGIdb（Drug-Gene Interaction Database）analyzed the possible gene-drug regulatory network of downstream genes,and selected those genes having gene-drug regulationship as hub genes.PPI method was used to analyze the regulatory relationship between hub genes.【Results】Part I:Compared with normol controls,circ METAP2 showed a significant downregulation trend in IPAH patients（over 2 fold-change,P=0.045）.ROC curve analysis showed that circ METAP2 had the best ability to distinguish IPAH from normal controls when the relative expression level of circ METAP2 were 0.715（sensitivity=72.2%,specificity=84.9%,AUC=0.842,P<0.001）in the total cohort,0.21（sensitivity=93.8%,specificity=57.1%,AUC=0.813,specificity=84.9%,P<0.001）in male cohort,and 0.71（sensitivity=80.0%,specificity=84.2%,AUC=0.860,P<0.001）in female cohort.Female IPAH patients were diagnosed at a younger age than male patients（38.7±11.8 years V.S.50.5±18.9 years,P=0.040）.Referring for sex hormone levels,the expression of TT in male IPAH patients was significantly higher than that in female patients[16.1（9.2,19.5）V.S.1.6（1.1,2.5）,nmol·L-1,P<0.001].the expression of E2 showed a lower but not significant expression trend[156.4（120.7,203.3）V.S.198.8（132.2,392.2）,pmol·L-1,P=0.054].The expression levels of P,LH and FSH between male and female patients showed no significane.In the total cohort,the relative expression of circ METAP2 was not significantly correlated with hemodynamic parameters.However,we found that the relative expression level of circ METAP2 was significantly negatively correlated with m PAP in female cohort（r=-0.296,P=0.045）,and the relative expression level of circ METAP2 was significantly positively correlated with m RAP in male cohort（R=0.402,P=0.046）.In the male cohort,univariate linear regression analysis showed that there was no significance between the expression level of sex hormones and the relative expression level of circ METAP2.In the female cohort,we found that the relative expression of circ METAP2 was significantly negatively correlated to the expression of E2（r=-0.352;95%CI:0.002,0.000;P=0.038）,and FSH（r=0.465;95%CI:0.159,0.015;P=0.019）.After multiple linear regression analysis,we found that decreased expression of circ METAP2 was an independent predictor of upregulated expression of FSH in female corhot（r=-0.363;95%CI:0.131,0.005;P=0.035）.According to the cut-off value of ROC analysis,there was no significant difference in event-free survival between patients with low expression of circ METAP2（<0.715）and patients with high expression of circ METAP2（≥0.715）in the total cohort（P=0.292）.In the male cohort,there was also no statistical difference in event-free survival between patients with low expression of circ METAP2（<0.21）and patients with high expression of circ METAP2（≥0.21）（P=0.288）.In the female cohort,we observed worse event-free survival in the group with low expression of circ METAP2（<0.71）than in the group with high expression of circ METAP2（≥0.71）（P=0.035）.Part II:Compared with PASMC cultured in normoxic（21%O2）state,circ METAP2 was significantly down-regulated in hypoxic（5%O2）PASMC.Compared with the saline treated group,circ METAP2 expression was significantly down-regulated in the lung tissues MCT-induced PH rats.Under normoxic（21%O2）state,there were no significant changes in proliferation,migration and death levels of PASMC after overexpressing circ METAP2.Under hypoxia（5%O2）state,overexpression of circ METAP2 significantly inhibited proliferation,and promoted migration and cell death of PASMC.Part III:Under hypoxia（5%O2）state,circ METAP2 is mainly located in the nucleus of PASMC.FISH combined with cell immunofluorescence confirmed that circ METAP2and SOX2 proteins could be overlapped at the microphysical level.Compared with PASMC under normoxic state,SOX2 protein was highly expressed in PASMC in hypoxic state.Under normoxic state,there was not significantly different expression of SOX2 protein among PASMCs from PBS treated group,Mock group and overexpressed circ METAP2 group.Under hypoxic state,the expression of SOX2protein in PASMC from the overexpressed circ METAP2 group was significantly lower than that of the PBS treated group and the Mock group.GTRD website obtained more than 1200 potential downstream targets of SOX2.GO clustering and KEGG enrichment analysis of these potential downstream targets showed that they were mainly enriched in RNA shearing and regulation.However,mitochondrial matrix（mitochondrial matrix）and mitochondrial inner membrance（mitochondrial inner membrance）from in cellular component（CC）terms suggested that it was involved in cellular respiration chain and related activities.DGIdb database found that213 genes of potential downstream targets of SOX2 had gene-drug regulatory relationships with existing drugs.Among them,8 genes were involved in the regulation of sex hormones and their metabolites,prostaglandin and fibroblasts growth factor,as the following:Progesterone is the drug with the most possible regulation genes,which can regulate CDK4,TP53,TDP1 and CP.Also,TDP1 may be regulated by testosterone,and CP may be regulated by estradiol.RECQL may be regulated by both estradiol and prostaglandin B1.ACTB,MAPK10 and ATF2 may be regulated by acetylene estradiol,fibroblast growth factor-1 and 2-methoxyestradiol,respectively.PPI analysis of these 213 genes as hub genes showed that the hub gene connection value（node＿dgree）of TP53,RPS27A,UBA52,UBC,CDK1,EPRS,BRCA1,NHP2 and GNB2L1 exceeded 100.TP53 was the only one of the overlapping between the eight genes with drug-gene regulatory relationship and the genes with over 100 value of node＿dgree.【CONCLUSIONS】Circ METAP2 showed a significant downregulated trend in the PBMC from IPAH patients,and and its relative expression level of 0.715 was the optimal threshold to distinguish IPAH from normal controls.In the total cohort,circ METAP2 expression was not significantly associated with hemodynamic parameters and event-free survival of IPAH,but it was different between male and female cohort.The relative expression of circ METAP2 was negatively correlated with m PAP in the female cohort,but positively correlated with m RAP in the male cohort.In the female cohort,the expression levels of E2 and FSH were negatively correlated with the relative expression level of circ METAP2,and the relative expression level of circ METAP2could independently negatively predict the expression of FSH.However,this was not observed in the male cohort.The lower circ METAP2 relative expression in female IPAH patients suggested a higher incidence of malignant events.Circ METAP2 was significantly downregulated in lung tissue from MCT-induced PH rats and hypoxic cultured PASMC.Overexpression of circ METAP2 can affect the proliferation,migration and cell death of hypoxic cultured PASMC.In the hypoxic state,circ METAP2 was mainly located in the nucleus of PASMC and overlapped with SOX2 protein at the microphysical level.Overexpression of circ METAP2 inhibited the expression of SOX2 in hypoxic cultured PASMC.Bioinformatics analysis showed that sex hormones might have relationship with the downstream of SOX2,and TP53might be the downstream target of SOX2. |
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