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MSC Promotes the Secretion of Exosomal miR-34a-5p and Improve Intestinal Barrier Function Through METTL3-Mediated Pre-miR-34A m6A Modification

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Abstract

Intestinal ischemia/reperfusion (I/R) injury (IIRI) is associated with high prevalence and mortality rate. Recently, mesenchymal stem cell (MSC) therapy attracted more attentions. However, the function and regulatory mechanism of MSC-derived exosomal miRNAs during IIRI remain largely uninvestigated. The in vitro and in vivo IIRI models were established. MSC were characterized by immunofluorescent staining and flow cytometry. Purified exosomes were characterized by transmission electron microscopy (TEM), flow cytometry, and western blot. The expression of key molecules was detected by western blot and qRT-PCR. CCK-8, TUNEL, and transepithelial electrical resistance (TER) assays were employed to assess cell viability, apoptosis, and intestinal integrity, respectively. Pre-miR-34A m6 modification was evaluated by methylated RNA immunoprecipitation (MeRIP)-qPCR. RNA pull-down and RIP were used to validate the direct association between pre-miR-34A and IGF2BP3. MSC-derived exosomal miR-34a-5p alleviated OGD/R-induced injury. In addition, MSC ameliorated OGD/R-induced injury through METTL3 pathway. Mechanistic study revealed that miR-34a-5p was modulated by METTL3/IGF2BP3-mediated m6A modification in MSC. The in vitro and in vivo functional experiments revealed that MSC secreted exosomal miR-34a-5p and ameliorated IIRI through METTL3/IGF2BP3-mediated m6A modification of pre-miR-34A. MSC promoted the secretion of exosomal miR-34a-5p and improved intestinal barrier function through METTL3/IGF2BP3-mediated pre-miR-34A m6A modification.

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Data Availability

All data generated or analyzed during this study are included in this article. The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Code Availability

Not Applicable.

Abbreviations

AMI:

Acute mesenteric ischemia

BMSC:

Bone derived mesenchymal stem cells

CM:

Conditioned media

DGCR8:

DiGeorge Critical Region 8

ELISA:

Enzyme-linked immunosorbent assay

H&E:

Hematoxylin and eosin

IBD:

Inflammatory bowel disease

IBS-D:

Diarrhea predominant irritable bowel syndrome

IECs:

Intestinal epithelial cells

IGF2BP3:

Insulin-like growth factor 2 mRNA binding protein 3

IF:

Immunofluorescence

I/R:

Ischemia/reperfusion

IIRI:

Intestinal ischemia reperfusion injury

m6A:

N6-methyladenosine

METTL3:

Methyltransferase-like 3

MeRIP:

Methylated RNA immunoprecipitation

miRNA:

MicroRNA

MSC:

mesenchymal stem cell

MSC-exo:

MSC-derived exosomes

ncRNAs:

non-coding RNAs

NEC:

necrotizing enterocolitis

OGD/R:

oxygen-glucose deprivation/reoxygenation

PFA:

paraformaldehyde

pre-miRNAs:

precursor miRNAs

pri-miRNAs:

primary microRNAs

RUNX2:

runt-related transcription factor 2

SRAMP:

sequence-based RNA adenosine methylation site predictor

TER:

transepithelial electrical resistance

TEM:

transmission electron microscopy

TJ:

tight junction

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Acknowledgements

We would like to give our sincere gratitude to the reviewers for their constructive comments. This work was supported by Yunnan Health Training Project of High-level Talents (No. D2017037), and Joint Project fund of Yunnan Provincial Science and Technology Department and Basic Research of Kunming Medical University (No. 202001AY070001-064). We would like to give our sincere gratitude to the reviewers for their constructive comments.

Funding

This work was supported by Yunnan Health Training Project of High-level Talents (No. D2017037), and Joint Project fund of Yunnan Provincial Science and Technology Department and Basic Research of Kunming Medical University (No. 202001AY070001-064).

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Authors and Affiliations

  1. Department of Gastrointestinal Surgery, Second Affiliated Hospital of Kunming Medical University, No.374, Dianmian Road, Kunming, 650101, Yunnan Province, People’s Republic of China

    Yi-Jun Li, Qing-Wen Xu, Cong-Hui Xu & Wei-Ming Li

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Contributions

YJL: conceptualization; writing-original draft; methodology; formal analysis;

QWX: conceptualization; writing—original draft; methodology; formal analysis;

CHX: data curation; resources; investigation; software;

WML: funding acquisition; project administration; visualization; Writing—review and editing.

All authors have read and approved the final version of this manuscript to be published.

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Correspondence to Wei-Ming Li.

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All animal studies were approved by the ethics committee of Kunming Medical University.

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The informed consent was obtained from the ethics committee of the Kunming Medical University.

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Not Applicable. This article does not contain any studies with human participants performed by any of the authors.

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Yi-Jun Li, Qing-Wen Xu and Wei-Ming Li are co-first authors.

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Li, YJ., Xu, QW., Xu, CH. et al. MSC Promotes the Secretion of Exosomal miR-34a-5p and Improve Intestinal Barrier Function Through METTL3-Mediated Pre-miR-34A m6A Modification. Mol Neurobiol 59, 5222–5235 (2022). https://doi.org/10.1007/s12035-022-02833-3

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