The mA methylation and expression profiles of mouse neural stem cells after hypoxia/reoxygenation.

BACKGROUND

Ischemia-reperfusion injury to the central nervous system often causes severe complications. The activation of endogenous neural stem cells (NSCs) is considered a promising therapeutic strategy for nerve repair. However, the specific biological processes and molecular mechanisms of NSC activation remain unclear, and the role of N6-methyladenosine (mA) methylation modification in this process has not been explored.

METHODS

NSCs were subjected to hypoxia/reoxygenation (H/R) to simulate ischemia-reperfusion in vivo. mA RNA methylation quantitative kit was used to measure the total RNA mA methylation level. Quantitative real-time PCR was used to detect methyltransferase and demethylase mRNA expression levels. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq) were conducted for NSCs in control and H/R groups, and the sequencing results were analyzed using bioinformatics. Finally, the migration ability of NSCs was identified by wound healing assays, and the proliferative capacity of NSCs was assessed using the cell counting kit-8, EdU assays and cell spheroidization assays.

RESULTS

Overall of mA modification level and Mettl14 mRNA expression increased in NSCs after H/R treatment. The mA methylation and expression profiles of mRNAs in NSCs after H/R are described for the first time. Through the joint analysis of MeRIP-seq and RNA-seq results, we verified the proliferation of NSCs after H/R, which was regulated by mA methylation modification. Seven hub genes were identified to play key roles in the regulatory process. Knockdown of Mettl14 significantly inhibited the proliferation of NSCs. In addition, separate analysis of the MeRIP-seq results suggested that mA methylation regulates cell migration and differentiation in ways other than affecting mRNA expression. Subsequent experiments confirmed the migration ability of NSCs was suppressed by knockdown of Mettl14.

CONCLUSION

The biological behaviors of NSCs after H/R are closely related to mA methylation of mRNAs, and Mettl14 was confirmed to be involved in cell proliferation and migration.