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. 2018 Oct:36:209-220.
doi: 10.1016/j.ebiom.2018.09.006. Epub 2018 Sep 20.

Cancer-associated fibroblasts contribute to oral cancer cells proliferation and metastasis via exosome-mediated paracrine miR-34a-5p

Yao-Yin Li  1 , Yi-Wei Tao  2 , Shuo Gao  3 , Pei Li  3 , Jian-Mao Zheng  4 , Si-En Zhang  5 , Jianfeng Liang  5 , Yuejiao Zhang  4
Affiliations

Cancer-associated fibroblasts contribute to oral cancer cells proliferation and metastasis via exosome-mediated paracrine miR-34a-5p

Yao-Yin Li et al. EBioMedicine. 2018 Oct.

Abstract

Background: Cancer-associated fibroblasts (CAFs) play an important role in regulating tumor progression by transferring exosomes to neighboring cells VSports手机版. Our aim was to clarify the role of microRNA encapsulated in the exosomes derived from CAFs in oral squamous cell carcinoma (OSCC). .

Methods: We examined the microRNA expression profiles of exosomes derived from CAFs and donor-matched normal fibroblasts (NFs) from patients with OSCC. We used confocal microscopy to examine the transportation of exosomal miR-34a-5p between CAFs and OSCC cells V体育安卓版. Next, luciferase reporter and its mutant plasmids were used to confirm direct target gene of miR-34a-5p. Phenotypic assays and in vivo tumor growth experiments were used to investigate the functional significance of exosomal miR-34a-5p. .

Findings: We found that the expression of miR-34a-5p in CAF-derived exosomes was significantly reduced, and fibroblasts could transfer exosomal miR-34a-5p to OSCC cells. In xenograft experiments, miR-34a-5p overexpression in CAFs could inhibit the tumorigenesis of OSCC cells. We further revealed that miR-34a-5p binds to its direct downstream target AXL to suppress OSCC cell proliferation and metastasis. Stable ectopic expression of AXL in OSCC cells overexpressing miR-34a-5p restored proliferation and motility abolished by the miRNA. The miR-34a-5p/AXL axis promoted OSCC progression via the AKT/GSK-3β/β-catenin signaling pathway, which could induce the epithelial-mesenchymal transition (EMT) to promote cancer cells metastasis. The miR-34a-5p/AXL axis enhanced nuclear translocation of β-catenin and then induced transcriptional upregulation of SNAIL, which in turn activated both MMP-2 and MMP-9. V体育ios版.

Interpretation: The miR-34a-5p/AXL axis confers aggressiveness in oral cancer cells through the AKT/GSK-3β/β-catenin/Snail signaling cascade and might represent a therapeutic target for OSCC. FUND: National Natural Science Foundation of China. VSports最新版本.

Keywords: AXL; CAFs; Exosomes; Oral squamous cell carcinoma; miR-34a-5p V体育平台登录. .

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Figures

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Graphical abstract
Fig. 1
Fig. 1
Characteristics of fibroblasts derived from patients with OSCC and isolation of exosomes. (A) Representative morphology of NFs and CAFs derived from patients with OSCC. Scale bar, 200 μm. (B) Western blotting analysis of the expression of CAF markers (α-SMA and FAP) and fibroblasts marker (vimentin) in isolated fibroblasts. (C) Immunofluorescence staining identification of CAFs using antibodies against vimentin, α-SMA, and FAP. Scale bar, 100 μm. (D) Transmission electron microscopy (TEM) showing exosomes isolated from NFs and CAFs-conditioned medium. Scale bar, 100 nm. (E) Western blotting analysis of the exosome marker CD63 and the Golgi matrix protein GM130 in exosome-enriched conditioned medium.
Fig. 2
Fig. 2
OSCC cells absorb miR-34a-5p-devoid exosomes derived from CAFs. (A) Heatmap diagram of differential miRNA expression profiles between CAFs and NFs-derived exosomes. Red = miRNAs with higher expression, blue = miRNAs with lower expression, and white = miRNAs with equal expression. (B) Differential miRNA expression profiles between CAFs and NFs-derived exosomes. Red = miRNAs with higher expression, green = miRNAs with lower expression, and gray = miRNAs with equal expression. (C) Real-time PCR analysis of miR-34a-5p expression in NFs and CAFs (n = 6). **P < .01. (D) Representative H&E and ISH staining of miR-34a-5p in OSCC and donor-matched normal tissue. Scale bar, 200 μm. (E) Real-time PCR analysis of miR-34a-5p expression in HOK, CAL27, SCC15, NFs, and CAFs, and 5 ng/ml TGF-β1-stimulated NFs and CAFs. *P < .05; **P < .01. (F) CAFs transfected with cy3-tagged miR-34a-5p (CAFs-miR-34a-5p-cy3) or with the miR-control, and GW4869-treated CAFs were indirectly co-cultured with OSCC cells for 24 h. Fluorescence microscopy was used to detect the α-tubulin (green) and cy3 (red) fluorescent signals in OSCC cells. Scale bar, 100 μm. (G) Exosomes were isolated from conditioned media derived from CAFs transfected with cy3-labeled miR-34a-5p (CAFs-miR-34a-5p-cy3 exo) or with miR-control (CAFs-miR-control exo) and 200 μg of exosomes were added to OSCC cells for 24 h. Fluorescence microscopy was used to detect the α-tubulin (green) and cy3 (red) fluorescent signals in OSCC cells. Scale bar, 100 μm. (H) Real-time PCR analysis of miR-34a-5p expression in OSCC cells treated with 200 μg of exosomes derived from CAFs (CAFs control exo) and from CAFs transfected with a lentiviral plasmid containing pre-miR-34a-5p (CAFs-miR-34a-5p exo) or with the miR-control (CAFs-miR-control exo). **P < .01.
Fig. 3
Fig. 3
MiR-34a-5p inhibits OSCC cell proliferation, colony formation, migration, and invasion both in vitro and in vivo. (A) The effect of miR-34a-5p overexpression on the growth rate of both CAL27 and SCC15 cells examined using the CCK-8 assay. *P < .05; **P < .01. (B) The effect of miR-34a-5p overexpression expression on colony formation in both CAL27 and OSCC15 cells examined using the colony formation assay. **P < .01. (C-D) The effect of miR-34a-5p overexpression on the migration (C) and invasion (D) of both CAL27 and OSCC15 cells examined using a Transwell assay. Scale bar, 200 μm. **P < .01. (E) SCC15 cells transfected with the lentiviral plasmid containing pre-miR-34a-5p or miR-control were injected subcutaneously into nude mice (n = 6). After 6 weeks, the mice were euthanized, and the tumors were excised. **P < .01. (F) CAL27 cells were coinjected with NFs, CAFs-miR-control, or CAF-miR-34a-5p into nude mice subcutaneously. After 6 weeks, the mice were euthanized, and the tumors were excised. **P < .01.
Fig. 4
Fig. 4
AXL is a direct target of miR-34a-5p in OSCC cells. (A) The target genes of miR-34a-5p were predicted using publicly available bioinformatics tools (TargetScan, miRWalk, miRTarBase, and miRanda). (B) The expression of AXL in both CAL27 and SCC15 cells was examined using western blotting after transfection with lenti-miR-control or lenti-miR-34a-5p. (C) AXL mRNA levels in CAL27 and SCC15 cells treated with exosomes derived from CAFs control, CAFs-miR-control or CAFs-miR-34a-5p were examined using real-time PCR. *P < .05; **P < .01. (D) Predicted miR-34a-5p binding sites in the 3′ UTR of wild-type (AXL-3′-UTR-WT) and mutant (AXL-3′-UTR-MUT) AXL sequences. (E) Luciferase reporter assays were performed 48 h after co-transfection of CAL27 and SCC15 cells with control or miR-34a-5p mimics and a luciferase vector encoding the wild-type or mutant AXL 3′ UTR region. *P < .05; **P < .01.
Fig. 5
Fig. 5
MiR-34a-5p inhibits OSCC cell proliferation, migration, and invasion by targeting AXL. (A) AXL expression in OSCC cells transfected with AXL was examined by western blotting. (B) Colony formation in both AXL-transfected CAL27 and SCC15 cells was examined using a colony formation assay. *P < .05; **P < .01. (C) The growth rate of AXL-transfected CAL27 and SCC15 cells was examined using the CCK-8 assay. *P < .05; **P < .01. (D-E) The migration (D) and invasion (E) of AXL-transfected CAL27 and SCC15 cells was examined using a Transwell assay. **P < .01. (F-H) Clone formation (F), migration (G), and invasion (H) of CAL27 cells transfected with lenti-miR-control, lenti-miR-34a-5p, lenti-miR-34a-5p mock and lenti-miR-34a-5p AXL-OE was examined using a colony formation assay and Transwell assay, respectively. **P < .01.
Fig. 6
Fig. 6
MiR-34a-5p exerts its functions by inhibiting EMT and MMP-2/9 activation in OSCC cells. (A-B) The activation of β-catenin in CAL27 cells transfected with lenti-miR-control, lenti-miR-34a-5p, lenti-miR-34a-5p mock, and lenti-miR-34a-5p AXL-OE was examined using western blotting (A) and immunofluorescence staining (B); scale bar, 100 μm. (C) The effect of lenti-miR-control, lenti-miR-34a-5p, lenti-miR-34a-5p mock, and lenti-miR-34a-5p AXL-OE transfection in CAL27 cells on AKT/GSK-3β/β-catenin signaling pathway was examined using western blotting. (D) The effect of two target-specific AXL siRNAs transfection and AXL overexpression in CAL27 cells on the AKT/GSK-3β/β-catenin signaling pathway was examined using western blotting. (E-F) The expression of Snail was detected in CAL27 cells transfected with si-AXL (E) or si-β-catenin (F) was examined using real-time PCR and western blotting, respectively. **P < .01. (G-H) The activation of MMP-2 and MMP-9 in CAL27 cells transfected with the Snail siRNA and the vector containing full-length Snail in CAL27 was examined by real-time PCR (G) and Gelatin zymography (H). *P < .05; **P < .01. (I) Representative H&E and immunohistochemical staining for AXL, Snail, β-catenin, E-cadherin, vimentin, MMP-2 and MMP-9 in miR-34a-5p overexpression in SCC15 cells xenograft tumor tissue; scale bar, 200 μm. (J) Schematic representation of the contribution of miR-34a-5p-devoid exosomes derived from CAFs to EMT in OSCC cells via AKT/GSK-3β/β-catenin/Snail signaling cascade.
Fig. S1
Fig. S1
NFs or CAFs do not form tumors in nude mice. (A) NFs (5 × 106) were injected into the left flank of six nude mice, and 5 × 106 CAFs were injected into the right flank of the same nude mice. (B) After 6 weeks, the mice were euthanized, and no tumor tissues were found.
Fig. S2
Fig. S2
AXL expression is positively associated with the motility of OSCC cells. (A-B) The expression of AXL in CAL27 and SCC15 cells was examined using western blotting (A) and real-time PCR (B). *P < .05. (C) The migration and invasion of CAL27 and SCC15 cells was examined using a Transwell assay. Scale bar, 200 μm. *P < .05.
Fig. S3
Fig. S3
MiR-34a-5p inhibits SCC15 cells colony formation, migration, and invasion by targeting AXL. (A-C) Clone formation (A), migration (B) and invasion (C) of SCC15 cells transfected with lenti-miR-control, lenti-miR-34a-5p, lenti-miR-34a-5p mock, and lenti-miR-34a-5p AXL-OE was examined using a colony formation assay and Transwell assay, respectively.
Fig. S4
Fig. S4
MiR-34a-5p exerts its functions by inhibiting EMT and MMP-2/9 activation in SCC15 cells. (A-B) The activation of β-catenin in SCC15 cells transfected with lenti-miR-control, lenti-miR-34a-5p, lenti-miR-34a-5p mock, and lenti-miR-34a-5p AXL-OE was examined using western blotting (A) and immunofluorescence staining (B); Scale bar, 100 μm. (C) The effect of lenti-miR-control, lenti-miR-34a-5p, lenti-miR-34a-5p mock, and lenti-miR-34a-5p AXL-OE transfection in SCC15 cells on the AKT/GSK-3β/β-catenin signaling pathway was examined using western blotting. (D) The effect of two target-specific AXL siRNAs transfection and AXL overexpression in SCC15 cells on the AKT/GSK-3β/β-catenin signaling pathway was examined using western blotting. (E-F) The expression of Snail was detected in SCC15 cells transfected with si-AXL (E) or si-β-catenin (F) using real-time PCR and western blotting, respectively. **P < .01. (G-H) The activation of MMP-2 and MMP-9 in SCC15 cells transfected with the Snail siRNA and a vector containing full-length Snail in SCC15 cells was examined using real-time PCR (G) and Gelatin zymography (H). *P < .05; **P < .01.
Fig. S5
Fig. S5
Snail mediates the expression of E-cadherin and vimentin in OSCC cells. (A-B) The expression of Snail, E-cadherin, and vimentin in Snail-silenced CAL27 cells (A) and Snail-overexpressed CAL27 cells (B) was examined using western blotting and real-time PCR, respectively. *P < .05; **P < .01. (C-D) The expression of Snail, E-cadherin, and vimentin in Snail-silenced SCC15 cells (C) and Snail-overexpressed SCC15 cells (D) was examined using western blotting and real-time PCR, respectively. **P < .01.
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