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Validated Impacts of N6-Methyladenosine Methylated mRNAs on Apoptosis and Angiogenesis in Myocardial Infarction Based on MeRIP-Seq Analysis

Objectives: N6-methyladenosine (m(6)A) is hypothesized to play a role in the regulation of pathogenesis of myocardial infarction (MI). This study was designed to compare m(6)A-tagged transcript profiles to identify mRNA-specific changes on pathophysiological variations after MI. Methods: N6-methylad...

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Autores principales: Zhang, Yingjie, Hua, Wenjie, Dang, Yini, Cheng, Yihui, Wang, Jiayue, Zhang, Xiu, Teng, Meiling, Wang, Shenrui, Zhang, Min, Kong, Zihao, Lu, Xiao, Zheng, Yu
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8831860/
https://www.ncbi.nlm.nih.gov/pubmed/35155564
http://dx.doi.org/10.3389/fmolb.2021.789923
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author Zhang, Yingjie
Hua, Wenjie
Dang, Yini
Cheng, Yihui
Wang, Jiayue
Zhang, Xiu
Teng, Meiling
Wang, Shenrui
Zhang, Min
Kong, Zihao
Lu, Xiao
Zheng, Yu
author_facet Zhang, Yingjie
Hua, Wenjie
Dang, Yini
Cheng, Yihui
Wang, Jiayue
Zhang, Xiu
Teng, Meiling
Wang, Shenrui
Zhang, Min
Kong, Zihao
Lu, Xiao
Zheng, Yu
author_sort Zhang, Yingjie
collection PubMed
description Objectives: N6-methyladenosine (m(6)A) is hypothesized to play a role in the regulation of pathogenesis of myocardial infarction (MI). This study was designed to compare m(6)A-tagged transcript profiles to identify mRNA-specific changes on pathophysiological variations after MI. Methods: N6-methyladenosine methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq) were interacted to select m(6)A-modified mRNAs with samples collected from sham operated and MI rat models. m(6)A methylation regulated mRNAs were interacted with apoptosis/angiogenesis related genes in GeneCards. Afterwards, MeRIP-quantitative real-time PCR (MeRIP-qRT-PCR) was performed to measure m(6)A methylation level of hub mRNAs. m(6)A methylation variation was tested under different oxygen concentration or hypoxic duration in H9c2 cells and HUVECs. In addition, Western blot and qRT-PCR were employed to detect expression of hub mRNAs and relevant protein level. Flow cytometry and Tunel assay were conducted to assess apoptotic level. CCK-8, EdU, and tube formation assay were performed to measure cell proliferation and tube formation ability. Results: Upregulation of Mettl3 was firstly observed in vivo and in vitro, followed by upregulation of m(6)A methylation level. A total of 567 significantly changed m(6)A methylation peaks were identified, including 276 upregulated and 291 downregulated peaks. A total of 576 mRNAs were upregulated and 78 were downregulated. According to combined analysis of MeRIP-seq and RNA-seq, we identified 26 significantly hypermethylated and downregulated mRNAs. Based on qRT-PCR and interactive analysis, Hadh, Kcnn1, and Tet1 were preliminarily identified as hub mRNAs associated with apoptosis/angiogenesis. MeRIP-qRT-PCR assay confirmed the results from MeRIP-seq. With the inhibition of Mettl3 in H9c2 cells and HUVECs, downregulated m(6)A methylation level of total RNA and upregulated expression of hub mRNAs were observed. Increased m(6)A level was verified in the gradient context in terms of prolonged hypoxic duration and decreased oxygen concentration. Under simulated hypoxia, roles of Kcnn1 and Tet1 in angiogenesis and Hadh, Tet1, and Kcnn1 in apoptosis were further confirmed with our validation experiments. Conclusion: Roles of m(6)A-modified mRNA transcripts in the context of MI were preliminarily verified. In the context of m(6)A methylation, three hub mRNAs were validated to impact the process of apoptosis/angiogenesis. Our study provided theoretical basis and innovative targets for treatment of MI and paved the way for future investigations aiming at exploring upstream epigenetic mechanisms of pathogenesis after MI.
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spelling pubmed-88318602022-02-12 Validated Impacts of N6-Methyladenosine Methylated mRNAs on Apoptosis and Angiogenesis in Myocardial Infarction Based on MeRIP-Seq Analysis Zhang, Yingjie Hua, Wenjie Dang, Yini Cheng, Yihui Wang, Jiayue Zhang, Xiu Teng, Meiling Wang, Shenrui Zhang, Min Kong, Zihao Lu, Xiao Zheng, Yu Front Mol Biosci Molecular Biosciences Objectives: N6-methyladenosine (m(6)A) is hypothesized to play a role in the regulation of pathogenesis of myocardial infarction (MI). This study was designed to compare m(6)A-tagged transcript profiles to identify mRNA-specific changes on pathophysiological variations after MI. Methods: N6-methyladenosine methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq) were interacted to select m(6)A-modified mRNAs with samples collected from sham operated and MI rat models. m(6)A methylation regulated mRNAs were interacted with apoptosis/angiogenesis related genes in GeneCards. Afterwards, MeRIP-quantitative real-time PCR (MeRIP-qRT-PCR) was performed to measure m(6)A methylation level of hub mRNAs. m(6)A methylation variation was tested under different oxygen concentration or hypoxic duration in H9c2 cells and HUVECs. In addition, Western blot and qRT-PCR were employed to detect expression of hub mRNAs and relevant protein level. Flow cytometry and Tunel assay were conducted to assess apoptotic level. CCK-8, EdU, and tube formation assay were performed to measure cell proliferation and tube formation ability. Results: Upregulation of Mettl3 was firstly observed in vivo and in vitro, followed by upregulation of m(6)A methylation level. A total of 567 significantly changed m(6)A methylation peaks were identified, including 276 upregulated and 291 downregulated peaks. A total of 576 mRNAs were upregulated and 78 were downregulated. According to combined analysis of MeRIP-seq and RNA-seq, we identified 26 significantly hypermethylated and downregulated mRNAs. Based on qRT-PCR and interactive analysis, Hadh, Kcnn1, and Tet1 were preliminarily identified as hub mRNAs associated with apoptosis/angiogenesis. MeRIP-qRT-PCR assay confirmed the results from MeRIP-seq. With the inhibition of Mettl3 in H9c2 cells and HUVECs, downregulated m(6)A methylation level of total RNA and upregulated expression of hub mRNAs were observed. Increased m(6)A level was verified in the gradient context in terms of prolonged hypoxic duration and decreased oxygen concentration. Under simulated hypoxia, roles of Kcnn1 and Tet1 in angiogenesis and Hadh, Tet1, and Kcnn1 in apoptosis were further confirmed with our validation experiments. Conclusion: Roles of m(6)A-modified mRNA transcripts in the context of MI were preliminarily verified. In the context of m(6)A methylation, three hub mRNAs were validated to impact the process of apoptosis/angiogenesis. Our study provided theoretical basis and innovative targets for treatment of MI and paved the way for future investigations aiming at exploring upstream epigenetic mechanisms of pathogenesis after MI. Frontiers Media S.A. 2022-01-28 /pmc/articles/PMC8831860/ /pubmed/35155564 http://dx.doi.org/10.3389/fmolb.2021.789923 Text en Copyright © 2022 Zhang, Hua, Dang, Cheng, Wang, Zhang, Teng, Wang, Zhang, Kong, Lu and Zheng. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Molecular Biosciences
Zhang, Yingjie
Hua, Wenjie
Dang, Yini
Cheng, Yihui
Wang, Jiayue
Zhang, Xiu
Teng, Meiling
Wang, Shenrui
Zhang, Min
Kong, Zihao
Lu, Xiao
Zheng, Yu
Validated Impacts of N6-Methyladenosine Methylated mRNAs on Apoptosis and Angiogenesis in Myocardial Infarction Based on MeRIP-Seq Analysis
title Validated Impacts of N6-Methyladenosine Methylated mRNAs on Apoptosis and Angiogenesis in Myocardial Infarction Based on MeRIP-Seq Analysis
title_full Validated Impacts of N6-Methyladenosine Methylated mRNAs on Apoptosis and Angiogenesis in Myocardial Infarction Based on MeRIP-Seq Analysis
title_fullStr Validated Impacts of N6-Methyladenosine Methylated mRNAs on Apoptosis and Angiogenesis in Myocardial Infarction Based on MeRIP-Seq Analysis
title_full_unstemmed Validated Impacts of N6-Methyladenosine Methylated mRNAs on Apoptosis and Angiogenesis in Myocardial Infarction Based on MeRIP-Seq Analysis
title_short Validated Impacts of N6-Methyladenosine Methylated mRNAs on Apoptosis and Angiogenesis in Myocardial Infarction Based on MeRIP-Seq Analysis
title_sort validated impacts of n6-methyladenosine methylated mrnas on apoptosis and angiogenesis in myocardial infarction based on merip-seq analysis
topic Molecular Biosciences
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8831860/
https://www.ncbi.nlm.nih.gov/pubmed/35155564
http://dx.doi.org/10.3389/fmolb.2021.789923
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