West China Journal of Stomatology ›› 2021, Vol. 39 ›› Issue (2): 136-142.doi: 10.7518/hxkq.2021.02.003

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microRNA-1 gene delivery mediated by exosomes suppresses CAL-27 cell proliferation

Wu Baoqin1,2(), Li Chunhui1,2, Zhang Menglian1, Nie Minhai1,2()   

  1. 1.Orofacial Reconstruction and Regeneration Laboratory, School of Stomatology of Southwest Medical University, Luzhou 646000, China
    2.Dept. of Periodontal and Oral Medicine, The Affiliated Stomatology Hospital of Southwest Medical University, Luzhou 646000, China
  • Received:2020-06-26 Revised:2020-09-09 Online:2021-04-01 Published:2021-04-09
  • Contact: Nie Minhai E-mail:wubaoqinasd@163.com;nieminhai@126.com
  • Supported by:
    Key Project of Education Department of Sichuan Province(17ZA0443);Project of Health and Family Planning Commission of Sichuan Province(16PJ535);Project of The Affiliated Stomatology Hospital of Southwest Medical University(201707)

Abstract: Objective

This study aims to construct endogenous exosomes abundantly loaded with miR-1 and investigate the role of exosome-mediated microRNA-1 (miR-1) delivery on CAL-27 cell proliferation.

Methods

Exosomes secreted by miR-1-overexpressing HEK293 cells (miR1-EXO) were purified via ultracentrifugation and subjected to transmission electron microscopy, nanoparticle analysis, Western blot analysis, and quantitative polymerase chain reaction (qPCR). CAL-27 cells were cocultured with exosomes secreted by HEK293 cells (CON-EXO) and miR1-EXO and equivalent phosphate buffer saline. The intracellular transport of exosomes was measured by using immunofluorescence, the expression of miR-1 and its target gene MET were investigated via qPCR, CAL-27 cell proliferation was measured through MTT assay, and cell cycle state was determined by applying flow cytometry.

Results

Electron microscopy revealed that miR1-EXO and CON-EXO were spherical or cup-shaped with an average diameter of approximately 110 nm. The well-known exosome markers CD9, Tsg101, and Alix were enriched. The expression of miR-1 in miR1-EXO was higher than that in CON-EXO (285.80±14.33 vs 1.00±0.06, P<0.000 1). After coculture with CAL-27 cells, miR1-EXO was internalized and unloaded miR-1 into CAL-27 cells. After coculture with miR1-EXO, the expression of miR-1 in CAL-27 cells was upregulated, whereas that of MET, the target gene of miR-1, was suppressed and the proliferation of CAL-27 cells was inhibited significantly. Normal oral keratinocyte cell proliferation was negligibly affected after coculture with miR1-EXO.

Conclusion

Exosomes secreted from miR1-EXO cells could load abundant miR-1. Exosomal miR-1 delivered into CAL-27 cells by using miR1-EXO suppressed the expression of MET mRNA and inhibited cell proliferation.

Key words: exosomes, microRNA-1, oral squamous cell carcinoma, cell proliferation

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