Study On The Role Of Hypoxia-induced Up-regulation Of DNA Methyltransferase 3A In Pulmonary Hypertension

Pulmonary hypertension(PAH),as a fatal disease,is a progressive vascular disease characterized by medial hypertrophy and hyperplasia of the pulmonary artery,extension of smooth muscle to non-muscular arterioles and small anterior capillary arterioles,resulting in complex pulmonary artery occlusion,progressive pulmonary arterial pressure and right heart failure.Pulmonary hypertension(PH)is divided into five groups,and PAH specifically refers to the first group of PH.In animal husbandry,yaks in the plateau area are prone to pulmonary hypertension when they migrate to different altitude areas.Ascites syndrome of broilers(ASB)is a hypoxia syndrome that leads to elevated pulmonary artery pressure and right heart failure in broilers.However,facing this deadly disease,there is no effective cure for it at present.Therefore,it is of great significance to study PAH both in human medicine and in veterinary medicine.Endothelial dysfunction is considered to be an important factor contributing to this vascular disease.Apoptosis resistance and excessive proliferation lead to pulmonary artery endothelial cells(PAECs)aggregation and formation of serious plexiform lesions,PAECs disorder caused by structural changes,changes in permeability,reduced nitric oxide synthesis ability and other factors are the inducement of endothelial dysfunction.PH patients with stenosis or even occlusion in pulmonary arteriole will cause oxygen supply capacity decline,lead to a state of hypoxia.The accumulation of hypoxia-inducible factors(HIFs)caused by hypoxia,which drive angiogenesis,endothelial cell proliferation,endothelial-mesenchymal transition(Endo-MT),redox state destruction and other physiological processes,plays an important role in the occurrence and development of PAH.Studies have shown that PH can lead to increased DNA methylation of superoxide dismutase 2(Sod2)promoter and inhibit its gene expression,thus affecting the production of eactive oxygen species(ROS).Whether hypoxia causes extensive genome-wide DNA methylation changes has not been fully studied.Based on the study of HIF2?,we analyzed the ability of HIF2?to differentiate PAH,studied the relationship between hypoxia and DNA methylation,screened DNA methyltransferase 3A(DNMT3A)as a target gene of hypoxia inducible factors,and further studied the effect of DNMT3A on endothelial cell function.?Objective?To explore the effects of hypoxia and hypoxia-inducible factors on DNA methylation in the process of PAH development,and to reveal the effect of DNMT3A on the function ofPAECs.?Methods?Using GEO transcriptome data,we first mined the differential values of gene expression profiles between wild-type(WT)and Hif2a knockdown(Hif2a-KD)clear cell Renal Cell Carcinoma(cc RCC)cell lines.We believe that the de-regulated gene between the wild type and Hif2a-KD cells is affected by HIF2?.One hand,by examining lung transcriptome data from PAH patients,we assessed the regulatory network of HIF2?in PAH pathogenesis to further determine the recognition ability of HIF2?mediated gene sets in human PAH subjects.On the other hand,using transcriptome data from peripheral blood mononuclear cells(PBMCs)from patients with PAH and healthy controls,we further validated the potential of the HIF2?mediated PBMCs gene sets as a possible diagnostic tool for PAH.To verify the ability of HIF2?mediated gene sets to identify PAH,we performed reverse validation with endothelial cell-specific knockout mice with spontaneous pulmonary hypertension.In order to reveal the effect of hypoxia on DNA methylation,we analyzed the DNA methylation spectrum of human trophoblast cells in GEO database and analyzed the DNA methylation levels under different oxygen concentrations to judge the possibility of hypoxia affecting DNA methylation.Through DNA methylation detection and transcriptome expression analysis of hPAECs and hPASMCs in PAH patients and controls,potential DNA methyltransferase candidate was searched.The expression and localization of DNMT3A were studied in SuHx-PH and HPH rat models,and the regulation of hypoxia and hypoxia-inducible factors on the expression of DNMT3A was studied in rPAECs and hPAECs.Using siRNA interference,we studied the effects of Dnmt3a-KD on rPAECs cell proliferation,apoptosis,autophagy,Endo-MT,ROS/NO production and other important physiological processes.we also explored the effect of hypoxia on NRF2 pathway which act as ROS regulator.?Results?Chapter 2:(1)The analysis of gene expression abundance in WT and Hif2a-KD Vhl deficiency cc RCCs obtained from GEO database showed that 19 GO terms were significantly correlated with gene downregulation in Hif2a-KD cells.These include"vascular development","angiogenesis","response to decreased oxygen levels","erythrocyte homeostasis",etc.All of these GO terms are closely related to the pathogenesis of PAH.(2)Analysis of lung transcriptome data sets from 9 controls and 8 PAH patients in GEO database showed that 14 of the 19 significantly down-regulated GO gene sets in Hif2a-KD cells overlapped with the up-regulated GO gene sets in lung tissues of PAH patients.Heat maps of gene set scores for these GO terms showed a clear separation between the control and the PAH patients,and we believe that these 14 overlapping GO terms are mediated by HIF2?in lung tissue.(3)Fourteen HIF2?mediated GO terms were tested in two published data sets of lung tissue transcriptome.Principal component analysis(PCA),the use of receiver operating characteristic curve(ROC)and the area under curve(AUC)all showed that HIF2?mediated GO terms had a strong ability to identify PAH.(4)PBMC transcriptome analysis of 10 controls and 8 PAH patients showed that HIF2?mediated GO gene sets could be used as biomarkers for PAH identification in high-risk populations.(5)Analysis of the correlation between hypoxia/normoxia and WT/Phd2^EC-/-gene expression fold changes(log2FC)in mouse lung tissues showed a significant positive correlation between their gene sets.The TGF?signaling pathway gene set score of Phd2^EC-/-mice significantly higher than that of WT mice,suggesting that activation of HIF2?and TGF?signaling pathways in endothelial cells of Phd2-deficient mice may lead to severe pulmonary hypertension.In the hif2a-KD cells,7 of the 19 significantly down-regulated GO terms overlapped with the up-regulated gene set in Phd2^EC-/-mice,suggesting that the HIF2?mediated gene sets also had strong recognition ability for experimental spontaneous PH models.Chapter 3:(1)Genome-wide DNA methylation of hPAECs and hPASMCs in PAH patients and non-PAH controls was detected to analyze gene promoter methylation levels.PCA showed that the overall DNA methylation patterns of hPAECs and hPASMCs were different,and the average level of promoter methylation of hPAECs was significantly higher than that of hPASMCs.GOBP/KEGG gene enrichment analysis showed that abnormal methylation genes in hPAECs of PAH patients were significantly associated with several gene sets in the pathogenesis of PH,such as"vascular development"and"positive regulation of cell adhesion".Similarly,some PH-related GOBP/KEGG GO terms,such as"calcium signaling pathway"and"leukocyte migration involved in inflammatory response",are also presenting in abnormal methylation gene sets of PAH hPASMCs.(2)Transcriptomic data of hPAECs and hPASMCs in PAH patients showed that compared with the control group,Dnmt1 and Dnmt3b were significantly down-regulated in PAH hPAECs,while Dnmt3a was significantly up-regulated in PAH hPAECs(FDR<5%).In contrast,we did not observe any significant differences in Dnmts gene expression between the control and PAH hPASMCs.Transcriptomic data of DNA methyltransferase genes in lung endothelial cells of WT and Hif2a-KO mice under hypoxia showed that Dnmt1 and Dnmt3b were significantly upregulated in Hif2a deficient cells,while Dnmt3a was significantly down-regulated,suggesting the potential transcriptional regulation of Dnmt3a by HIF2?.(3)Western blot analysis showed that compared with the control,DNMT3A was highly expressed in SuHx-PH rat lung tissues and primary endothelial cells isolated from HPH rat lung tissue.(4)Western blot analysis showed that hypoxia or DMOG up-regulated the expression of DNMT3A in rPAECs.Hypoxia also promoted the expression of DNMT3A in hPAECs.Dual-luciferase reporter assay showed that HIF1?/HIF2?binds to Dnmt3a promoter through hypoxia response element(HRE)to promote transcription.Chapter 4:(1)CCK,Edu and flow cytometric cell cycle analysis showed that Dnmt3a-KD reduced the viability and proliferation of rPAECs and blocked the cell cycle in G1 phase.Annexin V detection showed that Dnmt3a-KD promoted early apoptosis of rPAECs.Western blot analysis and immunofluorescence showed that Dnmt3a-KD promoted autophagy of rPAECs by up-regulating LC3B II and down-regulating p62.(2)The test of Endo-MT induced by TGF?in rPAECs showed that Dnmt3a-KD decreased the expression of?-SMA,SM22 which are important biomarkers in the process of Endo-MT.(3)NO detection results showed that Dnmt3a-KD enhanced NO production in hPAECs.Western blot results showed that Dnmt3a-KD down-regulated the expression of p-e NOS^T495,which relieved the inhibition of e NOS.(4)ROS detection results showed that Dnmt3a-KD reduced the production of ROS in rPAECs.Western blot results showed that Dnmt3a-KD up-regulated the expression of NRF2 downstream genes NQO1/HOMX1,which may play a important role in ROS clearance.(5)Western blot analysis showed that hypoxia decreased NRF2 expression,Hif2a-KD down-regulated NRF2 expression,and Nrf2-KD also down-regulated HIF2?expression under hypoxia.The results of dual-luciferase reporter assay showed that there was no transcriptional regulation between NRF2 and HIF2?through promoter.?Conclusions?HIF2?mediated GO gene sets have a great ability to distinguish PAH from controls.PAH caused differential methylation of hPAECs and hPASMC in gene promoters,and this differential methylation was partly caused by hypoxia-induciable factors mediated expression level change of DNMT3A.InPAECs,Dnmt3a-KD has been proved to inhibit the proliferation induced by hypoxia,promote apoptosis,autophagy and NO production,reduce ROS production,and inhibit the Endo-MT process induced by TGF?.These results suggest that Dnmt3a-KD provide a certain protective effect on endothelial cells during PAH development.