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N6-methyladenosine-modified MIB1 promotes stemness properties and peritoneal metastasis of gastric cancer cells by ubiquitinating DDX3X

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Abstract

Background

Peritoneal metastasis (PM), one of the most typical forms of metastasis in advanced gastric cancer (GC), indicates a poor prognosis. Exploring the potential molecular mechanism of PM is urgently necessary, as it has not been well studied. E3 ubiquitin ligase has been widely established to exert a biological function in various cancers, but its mechanism of action in GC with PM remains unknown.

Methods

The effect of MIB1 on PM of GC was confirmed in vitro and in vivo. Co-immunoprecipitation (Co-IP) and mass spectrometry demonstrated the association between MIB1 and DDX3X. Western blot, flow cytometry and immunofluorescence determined that DDX3X was ubiquitylated by MIB1 and promoted stemness. We further confirmed that METTL3 promoted the up-regulation of MIB1 by RNA immunoprecipitation (RIP), luciferase reporter assay and other experiments.

Results

We observed that the E3 ubiquitin ligase Mind bomb 1 (MIB1) was highly expressed in PMs, and patients with PM with high MIB1 expression showed a worse prognosis than those with low MIB1 expression. Mechanistically, our study demonstrated that the E3 ubiquitin ligase MIB1 promoted epithelial-mesenchymal transition (EMT) progression and stemness in GC cells by degrading DDX3X. In addition, METTL3 mediated m6A modification to stabilize MIB1, which required the m6A reader IGF2BP2.

Conclusions

Our study elucidated the specific molecular mechanism by which MIB1 promotes PM of GC, and suggested that targeting the METTL3-MIB1-DDX3X axis may be a promising therapeutic strategy for GC with PM.

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Data availability

The datasets generated during and analysed during the current study are not publicly available due other members of our group need to use RNA-seq data but are available from the corresponding author on reasonable request.

Abbreviations

GC:

Gastric cancer

PM:

Peritoneal metastasis

MIB1:

Mind bomb 1

DDX3X:

DEAD-box helicase 3 X-linked

EMT:

Epithelial-mesenchymal transition

LC/MS:

Liquid chromatography/mass spectrometry

Co-IP:

Co-Immunoprecipitation

RIP:

RNA-binding protein immunoprecipitation

MeRIP:

Methylated RNA Immunoprecipitation

qRT-PCR:

Quantitative real-time polymerase chain reaction

IHC:

Immunohistochemistry

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Acknowledgements

This study was supported by the Postgraduate Research & Practice Innovation Program of Jiangsu Province (SJCX21_0622), Jiangsu Province Capability Improvement Project through Science, Technology and Education (Jiangsu Provincial Medical Key Discipline, ZDXK202222). We would like to thank the Core Facility of the First Affiliated Hospital of Nanjing Medical University for its help in the detection of experimental samples.

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Author notes

  1. Peng Xu, Kanghui Liu, Shansong Huang and Jialun Lv have contributed equally to this work.

Authors and Affiliations

  1. Department of General Surgery, the First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu Province, China

    Peng Xu, Kanghui Liu, Shansong Huang, Jialun Lv, Zhengyuan Yan, Han Ge, Quan Cheng, Zetian Chen, Peicheng Ji, Yawei Qian, Bowen Li, Hao Xu, Li Yang, Zekuan Xu & Diancai Zhang

  2. Department of Surgery, Nanjing Lishui People’s Hospital, Nanjing, 211200, China

    Zhengyuan Yan

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  1. Peng Xu
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Contributions

DZ conceptualized and supervised the research. PX, KL, SH and JL designed and performed most experiments. ZY, HG, QC, ZC performed animal experiments. PJ, BL, HX, LY were engaged in biostatistics and bioinformatics analysis. ZX is responsible for the collection of tissues and follow-up of patients.

Corresponding author

Correspondence to Diancai Zhang.

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All procedures conformed to the ethical standards of the responsible committee on human experimentation and to the Helsinki Declaration of 1964 and later versions. Informed consent or the equivalent consent was obtained from all patients included in the study.

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Xu, P., Liu, K., Huang, S. et al. N6-methyladenosine-modified MIB1 promotes stemness properties and peritoneal metastasis of gastric cancer cells by ubiquitinating DDX3X. Gastric Cancer 27, 275–291 (2024). https://doi.org/10.1007/s10120-023-01463-5

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