Integrated Analysis Of M~6A MRNA Methylation In Rats With Monocrotaline-induced Pulmonary Arterial Hypertension

Background:N6-methyladenosine（m⁶A）modification is one of the most common chemical modifications of eukaryotic mRNAs and exerts important effects on mRNA stability,splicing,and translation.Recently,m⁶A has been increasingly recognized to play an important role in tumors and cardiovascular disease,and regulation of m⁶A modification may become a new treatment strategy for the disease.However,the changes of m⁶A mRNA and m⁶A-related enzymes in pulmonary arterial hypertension（PAH）have been little studied.The aim of this study was to investigate the changes of mRNA m⁶A in the rats lung tissue of PAH induced by monocrotaline（MCT）,and to provide a new idea for the pathogenesis of PAH.Methods:（1）Animal model construction:twenty male SD rats were randomly divided into control group（Con）and monocrotaline pulmonary arterial hypertension group（MCT-PAH）.Con group was intraperitoneal injection of the same amount of saline,MCT-PAH group was treated with intraperitoneal injection of monocrotaline（MCT）once,in dose of 60mg·kg^-1.（2）Pulmonary artery acceleration time（PAAT）,right ventricular internal diameter（RVID）and tricuspid annulus systolic displacement（TAPSE）were measured by doppler echocardiography;right ventricular systolic pressure（RSVP）was measured by cardiac catheterization;HE staining was used to detect pulmonary vascular changes.（3）Methylation sequencing was performed to detect the m⁶A modification level in lung tissues of rats in Con group and MCT-PAH group.Gene ontology（GO）was used to analyze the molecular function,cell composition and biological process.Kyoto Encyclopedia of Genes and Genomes（KEGG）analysis was used to predict the possible signaling pathways involved in the involvement of differential m⁶A modified genes in PAH.（4）Western blot was used to detect the expression of METTL3,METTL14,WTAP,VIRMA,RBM15,FTO,ALKBH5,YTHDFs,YTHDCs and IGF2BPs proteinases in rats lung tissues.（5）The expression and distribution of FTO,ALKBH5,METTL3 and YTHDF1 in pulmonary vessels were detected by immunofluorescence combined with laser confocal scanning.Results:（1）Compared with Con group,PAAT shortened,RVID increased,TAPSE decreased and RV/（LV+S）ratio increased in the MCT-PAH group.Right heart catheterization showed the RSVP significantly increased in MCT-PAH group;HE staining indicated that the pulmonary artery vessel wall was significantly thickened and vascular remodeling was present.（2）A differential pattern of m⁶A abundance was observed mainly up-methylated in the lung tissues of rats with MCT-induced PAH.GO and KEGG pathway analysis showed that m⁶A methylation of NF-κB,COL1A1,LDHA,PKM and other genes were up-regulated in PAH,and the up-regulated genes were enriched in the immune response,extracellular matrix receptor interaction,glycolysis,PI3K/Akt signaling pathways.（3）Compared with Con group,the expressions of FTO and ALKBH5 in lung tissue of MCT-PAH rats were decreased,while the expression of METTL3 and YTHDF1 were increased,and other methylated modified proteins had no significant changes.（4）Immunofluorescence combined with laser confocal scanning indicated that the expression of FTO in the pulmonary vessels of rats in the MCT-PAH group was decreased,the expression of YTHDF1 was increased,while the expression of ALKBH5 and METTL3 showed no significant difference in the pulmonary vessels.Conclusions:（1）The level of m⁶A methylation was increased in the lung tissues of rats with pulmonary arterial hypertension,and the methylation up-regulated genes were enriched in the extracellular matrix receptor interactions,glycolysis and PI3K/Akt signaling pathways.（2）The expressions of FTO and ALKBH5 in MCT-PAH rats were decreased,while the expressions of METTL3 and YTHDF1 were increased.Meanwhile,the expression of FTO decreased and the expression of YTHDF1 increased in pulmonary vessels.（3）FTO and YTHDF1 may play a leading role in mRNA m⁶A of MCT-PAH rats,and may be involved in the development of PAH through up-regulation of NF-κB,COL1A1,LDHA,PKM and other gene methylation levels,or immune response,glycolysis,PI3K/Akt and other signaling pathways.