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Co-immunoprecipitation Assay Using Endogenous Nuclear Proteins from Cells Cultured Under Hypoxic Conditions
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Co-immunoprecipitation Assay Using Endogenous Nuclear Proteins from Cells Cultured Under Hypoxic Conditions

Published on: August 2, 2018

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N6-methyladenosine methyltransferase plays a role in hypoxic preconditioning partially through the interaction with

Yamin Su1, Rongfeng Xu1, Rui Zhang1

  • 1Department of Cardiology, Zhongda Hospital, School of Medicine, Southeast University, Nanjing 210009, China.

Acta Biochimica Et Biophysica Sinica
|November 16, 2020
PubMed

Insights

Hypoxic preconditioning (HPC) protects cells from injury by increasing N6-methyladenosine (m6A) levels. This involves methyltransferase-like 3 (METTL3), methyltransferase-like 14 (METTL14), and H19 long noncoding RNA (lncRNA), offering potential therapeutic targets.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Epigenetics

Background:

  • N6-methyladenosine (m6A) is a crucial RNA modification with diverse biological roles.
  • The function of m6A in hypoxic preconditioning (HPC) remains largely unexplored.
  • HPC confers cellular protection against various stresses.

Purpose of the Study:

  • To investigate the role of m6A methylation in HPC-induced protection of H9c2 cells.
  • To identify key molecular players involved in m6A regulation during HPC.
  • To explore the potential of m6A-related factors as therapeutic targets in cardiac rehabilitation.

Main Methods:

  • H9c2 cells were subjected to hypoxic preconditioning (HPC) and H2O2-induced injury.
  • Expression levels of m6A, METTL3, METTL14, and H19 were quantified.
  • Knockdown and overexpression strategies were employed for METTL3, METTL14, and H19.
  • Methylated RNA immunoprecipitation (mRIP) and RNA binding protein immunoprecipitation (RIP) assays were performed.

Main Results:

  • HPC treatment enhanced H9c2 cell viability and reduced apoptosis.
  • HPC upregulated total RNA m6A levels, METTL3, METTL14, and H19 expression.
  • Knockdown of METTL3 or METTL14 diminished HPC-induced protection and H19 upregulation.
  • METTL3 and METTL14 directly bind to H19, regulating its m6A modification.

Conclusions:

  • A novel posttranscriptional regulatory mechanism involving m6A, METTL3, METTL14, and H19 is identified in HPC.
  • METTL3, METTL14, and H19 are critical mediators of HPC-induced cellular protection.
  • These factors represent potential biomarkers and therapeutic targets for HPC-related cardiac rehabilitation.
Keywords:
N6-methyladenosinehypoxic preconditioninglncRNA H19methyltransferase like 14methyltransferase like 3

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