Preliminary Study On The Study On The Role Of MRNA M~6A Modification In Idopathic Pulmonary Arterial Hypertension

Objective and significance:N6-methyladenosine is a common mRNA post-translational modification in eukaryotes,which plays a role in stem cell reprogramming,cell proliferation and diseases by dynamically regulating the splicing stability and translation efficiency of mRNA through m⁶A-related enzymes.More and more studies have shown that mRNA m⁶A disorder may be one of the sign of cardiovascular disease.However,the change of mRNA m⁶A modification in peripheral blood of idiopathic pulmonary hypertension remain unclear.The study was first to construct the transcriptom-wide map of m⁶A mRNAs in the peripheral blood of IPAH patients.10 hub genes cotaining differentially m⁶A methylated sites were selected by RNA-seq and bioinformatics analysis which associated with the development of idiopathic pulmonary hypertension.In addition,the change of m⁶A-related enzyme in tissues and cells were investigated by constructing hypoxia-induced pulmonary hypertension rat model and hypoxia-induced pulmonary artery smooth muscle cell model,thus providing a theoretical basis for further discussion and research on mRNA m⁶A modification in idiopathic pulmonary hypertension.Methods:In the first part,the peripheral blood of 6 normal adults and 6 patients with idiopathic pulmonary hypertension were collected.Methylated RNA Immunoprecipation Sequening（Me RIP-seq）was used to detect the differentially methylated m⁶A sites（DMMSs）.The differential expressed genes containing DMMSs were screened by RNA-seq and Me RIP-seq.The second part was animal model construction:（1）Twelve male Sprague Dawley（SD）rats with a body weight of 180～200g were randomly selected and divided into Normoxia group and Hypoxia group.（2）Right ventricular diameter（RVID）,tricuspid systolic displacement（TAPSE）,pulmonary valve acceleration time（PAAT）,right ventricular catheterization were measured by Doppler echocardiography,RVSP were detected by the right cardiac catheterizatio and HE staining was used to identify the hypoxia-induced pulmonary hypertension（HPH）rats model.（3）Western blot was used to detect the protein expression of METTL3,FTO,ALKBH5,YTHDF1,YTHDF2,IGF2BP1,IGF2BP2 in the lung tissues of Normoxia and Hypoxia groups.The third part was in vitro cell assay:（1）Primary PASMCs were extracted by tissue mass method,andα-SMA was identified by cellular immunofluorescence assay.（2）They were divided into two groups,the normoxic group（21%O₂）and the hypoxic group（1%O₂）.The normoxic group was put into an incubator（37℃,5%CO₂,21%O₂）while the hypoxic group was put into a hypoxic incubator（37℃,5%CO₂,1%O₂,94%N₂）for 24h.（3）Western blot was used to detect the protein expression of PCNA,and the construction of hypoxic induced PASMCs cell model was verified.（3）The protein expression of METTL3,FTO,ALKBH5,YTHDF1,YTHDF2,IGF2BP1,IGF2BP2 was detected by Western blot.Results:The first part（1）There were 2170 differentially downmethylaed m⁶A sites and 506 differentially upmethylated m⁶A sites in peripheral blood of IPAH patients,which were mainly distributed in CDS.（2）388 differentially methylated m⁶A sites and differentially expression genese were select by Me RIP-seq and RNA-seq.Ten hub target genes were screened out by Cytoscape software which were TP53,SMAD3,FOXO3,JUN,RPS27A,AGO3,CSNK1E,RUNX3,PIAS4,CBX4,the first three genes have been confirmed to be associated with pulmonary hypertension.（3）The RNA expression value of JUN,RPS27A,AGO3,CSNK1E,RUNX3,CBX4 in peripheral blood of IPAH patients was lower than normal（P<0.05）.The second part（1）Compared with the Normoxia group,TAPSE decreased,PAAT shorted and RVID increased in the Hypoxia group（P<0.05）.（2）The pulmonary systolic pressure measured by right heart catheter in the Hypoxia group was higher than that in the Normoxia group（P<0.001）.（3）Compared with Normoxia group,the mediums of pulmonary vessels in the Hypoxia group were significantly thickened（P<0.05）and the lumen was significantly narrowed,in addition,the proportion of FM vessels（P<0.01）and PM vessels（P<0.001）in the hypoxia group were increased,while the proportion of NM vessels was decreased compared with Normxia group（P<0.001）.（4）Compared with the Normoxia group,the protein expression of METTL3,FTO,ALKBH5,YTHDF1,YTHDF2,IGF2BP1,IGF2BP2 in the lung tissue of Hypoxia group were increased,and the protein expression of ALKBH5 and IGF2BP1 were decreased（P<0.05）.The third part（1）the extracted primary cells were identified as smooth muscle cells by cellular immunofluorescence.（2）Western blot showed that the protein expression of PCNA,METTL3,YTHDF1 was increased compared with 21%O2 group,the protein expression of IGF2BP1 was decreased（P<0.05）.（3）YTHDF1 and IGF2BP1 was predicted to be capable of binding to TP53,SMAD3,FOXO3,JUN,RPS27A,AGO3,CSNK1E,RUNX3,PIAS4,CBX4 by m6A2Target website.Conclusion:（1）Our study was the first to construct the transcriptome-wide map of m⁶A mRNA identified in the peripheral blood of idiopathic pulmonary hypertension patients and ten hub genes which play a role in the development of idiopathic pulmonary hypertension were selected out.The m⁶A level of TP53,SMAD3,FOXO3,JUN,AGO3,CSNK1E,RUNX3,PIAS4,CBX4 was decreased and the m⁶A level of RPS27A was increased.（2）The protein expression of YTHDF1 was upregulated and the protein expression of IGF2BP1 was downregulated in the lung tissues of hypoxic pulmonary hypertension rat model and hypoxic PASMCs.（3）IGF2BP1 and YTHDF1 binding with these differentially methylated genes may change the mRNA stability or protein expression to promote the proliferation of PASMCs and pulmonary vascular remodeling.