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. 2023 Jun 7;45(6):4936-4947.
doi: 10.3390/cimb45060314.

M2 Macrophages-Derived Exosomal miRNA-23a-3p Promotes the Progression of Oral Squamous Cell Carcinoma by Targeting PTEN

Jun Li  1   2   3 , Yongjie Bao  1   2   3 , Sisi Peng  1   2   3 , Chao Jiang  1   2   3 , Luying Zhu  1   2   3 , Sihai Zou  1   2   3 , Jie Xu  1   2   3 , Yong Li  1   2   3
Affiliations

M2 Macrophages-Derived Exosomal miRNA-23a-3p Promotes the Progression of Oral Squamous Cell Carcinoma by Targeting PTEN

Jun Li et al. Curr Issues Mol Biol. .

Abstract

Exosomes from tumor cells and immune cells regulate the tumor microenvironment through the biomolecules or microRNAs (miRNAs) they carry. This research aims to investigate the role of miRNA in exosomes derived from tumor-associated macrophages (TAMs) in the progression of oral squamous cell carcinoma (OSCC). RT-qPCR and Western blotting assays were used to determine the expression of genes and proteins in OSCC cells. CCK-8, Scratch assay and invasion-related proteins were utilized to detect the malignant progression of tumor cells. High-throughput sequencing predicted differentially expressed miRNAs in exosomes secreted by M0 and M2 macrophages. Compared with exosomes from M0 macrophages, exosomes from M2 macrophages led to enhanced proliferation and invasion of OSCC cells and inhibited their apoptosis. High-throughput sequencing results show that miR-23a-3p is differentially expressed in exosomes from M0 and M2 macrophages. MiRNA target gene database predicts that phosphatase and tensin homolog (PTEN) are target genes of miR-23a-3p. Further studies revealed that transfection of miR-23a-3p mimics inhibited PTEN expression in vivo and in vitro and promoted the malignant progression of OSCC cells, which was reversed by miR-23a-3p inhibitors. MiR-23a-3p in exosomes derived from M2 macrophages promotes malignant progression of OSCC. PTEN is a potential intracellular target of miR-23a-3p. MiR-23a-3p, an M2 macrophage-associated exosome, is a promising target for the future treatment of OSCC.

Keywords: M2 macrophages; MiR-23a-3p; OSCC; PTEN; exosome.

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Conflict of interest statement

The authors declare that the research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Extraction and identification of M0 and M2 exosomes. After 24 h of PMA (100 ng/mL) stimulation, THP-1 cell lines were treated with IL-4 (20 ng/mL) for 24 h. (A–C) Macrophage surface markers were detected. (A) Morphology of macrophages was observed under microscope. (B) RT-PCR analysis, (C) Western blot analysis. (D) Exosomes were obtained from cell supernatant via differential centrifugation. (E–G) Identification of exosomes: (E) Identification of exosome structure under an electron microscope (20,000×). (F) Detection of the size and number of exosomes via nanoparticle tracking analysis (dilution factor: 1:1000). (G) Detection of exosome markers with Western blot analysis. Data represent the mean ± standard errors of three separate experiments. * p < 0.05, ** p < 0.01, ns: not significant according to one-way ANOVA (versus M0 group).
Figure 2
Figure 2
M2 macrophage exosomes promote the progression of OSCC cells (A) CCK-8 assays were performed to determine cell viability at 0, 24, 48, and 72 h after cell treatment. (B,C) Effect of M2 macrophage-derived exosomes on apoptosis-related cytokine in OSCC cells. (B) RT-PCR analysis. (C) Western blot analysis. (D) The migration activity of Cal-27 cells was measured via wound healing assay. The wound area was calculated at 0 h, 24 h, and 48 h after cell treatment. (E,F) The expression of EMT related protein in Cal-27 cells is affected by M2 macrophage-derived exosomes. (E) RT-PCR analysis. (F) Western blot analysis. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns: not significant by one-way ANOVA (versus control group).
Figure 3
Figure 3
MiR-23a-3p promotes malignant progression of OSCC cells (A) In silico analysis of regulatory miRNAs related to M0 and M2 macrophage exosomes. Cal-27 was treated with M2 macrophage exosomes or miR-23a-3p mimics after stable transfection with miR-23a-3p inhibitor. (B) RT-PCR analysis of miR-23a-3p in Cal-27 cells after transfection. (C) CCK-8 assays were performed to determine cell viability at 0, 24, 48, and 72 h after cell treatment. (D,E) RT-PCR and Western blot were used to detect the levels of apoptosis-related factors. (F) The motility of Cal-27 cells was determined via wound healing assays. EMT-associated proteins in Cal-27 cells were detected via Western blot (G) and RT-PCR (H). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns: not significant according to one-way ANOVA.
Figure 4
Figure 4
MiR-23a-3p targets PTEN (A) The three circles represent the target gene of miR-23a-3p in the three miRNA databases, and the middle part represents the intersection of the three datasets. (B) KEGG analysis of 78 target genes. (C) The predicted binding sites between miR-23a-3p and PTEN genes were predicted in the miRBD database. (D,E) RT-PCR and Western blot analysis of miR-23a-3p in Cal-27 cells. (F) Quantitative analysis. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 according to one-way ANOVA.
Figure 5
Figure 5
MiR-23a-3p promotes tumor growth in vivo. (A) The tumor. (B) Tumor weight. (C) Tumor volume. (D) Tunel-positive cells (green fluorescence) and their proportion. The nucleus is stained with DAPI (blue). (E) Immunohistochemical analyses of Ki67 in tumors. (F,G) Expression level of PTEN in tumor tissues. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns: not significant according to one-way ANOVA.
Figure 6
Figure 6
Graphical illustration of the effect of miRNA-23a-3p on OSCC cells.
See this image and copyright information in PMC

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