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ABSTRACT
Idiopathic pulmonary arterial hypertension (IPAH) is a serious and fatal disease. Recently, m6A has been reported to play an important role in the lungs of IPAH patients and experimental pulmonary hypertension models. However, the meaning of m6A mRNAs in the peripheral blood of IPAH patients remains largely unexplored. We aimed to construct a transcriptome-wide map of m6A mRNAs in the peripheral blood of IPAH patients. M6A RNA Methylation Quantification Kit was utilized to measure the total m6A levels in the peripheral blood of IPAH patients. A combination of MeRIP-seq, RNA-seq and bioinformatics analysis was utilized to select m6A-modified hub genes of IPAH. MeRIP-qPCR and RT-qPCR were used to measure the m6A levels and mRNA levels of TP53, RPS27A, SMAD3 and FoxO3 in IPAH patients. Western blot was performed to assess the protein levels of m6A related regulators and m6A related genes in experimental PH animal models, hypoxia-treated and PDGF-BB induced PASMCs. We found that the total m6A levels were increased in peripheral blood of IPAH patients and verified that m6A levels of RPS27A and SMAD3 were significantly elevated and m6A levels of TP53 and FoxO3 were significantly reduced. The mRNA or protein levels of RPS27A, SMAD3, TP53 and FoxO3 were changed in human blood samples, experimental PH animal models and PDGF-BB induced PASMCs. Moreover, METTL3 and YTHDF1 were increased in the hypoxia induced pulmonary hypertension rat model, hypoxia-treated and PDGF-BB induced PASMCs. These finding suggested that m6A may play an important role in IPAH.
Acknowledgments
We thanked all subjects who participated in this study. We thank Cloud-Seq Biotech Ltd. Co. (Shanghai, China) for the MeRIP-Seq service and the subsequent bioinformatics analysis.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Authors’ contributions
TH, YHZ, YBX and YCD contributed to design the research; TH, YHZ and HQF carried out the experiments; TH, YHZ, YY, WFL, JQL, FY and WJC analyzed the test data and made the figures; TH and YHZ drafted the manuscript; YBX helped polish the manuscript. All authors read and approved the final manuscript.
Availability of data and materials
The datasets generated and analyzed during the current study are available in the Sequence Read Archive (SRA) repository with accession number SRR19118209, SRR19118210, SRR19118211, SRR19118212, SRR19118213, https://www.ncbi.nlm.nih.gov/sra.
Ethics approval and consent to participate
The protocol of this research was submitted to and approved by the Institutional Animal Care and Ethics Committee of Hunan Children’s Hospital, China (permit number: HCHLL-2020-44) and written informed consent was obtained from all subjects and/or their legal guardian(s). All experimental methods related to animals were approved by the Institutional Animal Care and Use Committee of Hunan Children’s Hospital. We confirmed that all methods were performed in accordance with the relevant guidelines and regulations and that the study was reported in accordance with ARRIVE guidelines.