Cancer-associated fibroblast-released extracellular vesicles carrying miR-199a-5p induces the progression of​ gastric cancer through regulation of FKBP5-mediated AKT1/mTORC1 signaling pathway

Abstract

Accumulating evidence has unfolded the significance of extracellular vesicles (EVs) in diseases and cancers. Here, we attempted to discuss the role of cancer-associated fibroblasts (CAFs)-derived EVs containing miR-199a-5p in gastric tumorigenesis. Upregulated miR-199a-5p was first identified in cancer cells. Then, we selected CAFs for isolation of EVs which were co-cultured with AGS cells. We observed successful delivery of miR-199a-5p via CAF-derived EVs. Besides, miR-199a-5p promoted malignant properties of AGS cells. Moreover, miR-199a-5p downregulated FKBP5, leading to upregulated phosphorylation level of AKT1, which promoted the malignant phenotypes of AGS cells by activating mammalian target of rapamycin complex 1(mTORC1). Exosomal miR-199a-5p from CAFs promoted gastric tumorigenesis in vivo. Our findings point toward the critical role of CAFs-derived EVs carrying miR-199a-5p in gastric cancer progression.

Keywords: AKT1; Cancer-associated fibroblasts; FKBP5; extracellular vesicles; gastric cancer; mTORC1; microRNA-199a-5p.

Figure 1.

CAFs-EVs promoted the occurrence and development of gastric cancer through miR-199a-5p. (a) Morphology of CAFs-EVs was observed by transmission electron microscope (left), and the concentrations of CAFs-EVs were estimated with a BCA protein assay kit (right). (b) After co-cultured with CAFs, RT-qPCR was used to detect the expression of miR-199a-5p in human normal gastric epithelial cell line GES-1 and gastric cancer cell lines HGC-27, NCI-N87, AGS and SNU-5. (c) The expression of miR-199a-5p was determined in AGS cells co-cultured with CAFs or CAFs-EVs by RT-qPCR. (d) The expression of miR-199a-5p was detected by RT-qPCR after AGS cells were co-cultured with CAFs-EVs carrying miR-199a-5p inhibitor or mimic. (e) CD63-labeled CAFs-EVs were co-cultured with AGS cells after transfection with Cy3-miR-199a-5p-mimic, and co-localization of CD63 and Cy3 in AGS cells was detected by confocal microscopy (1000 ×). (f) AGS cells were co-cultured with CAFs-EVs after transfection with miR-199a-5p inhibitor, and then the proliferation of AGS cells was detected by EdU staining. (g) AGS cells were co-cultured with CAFs-EVs after transfection with miR-199a-5p inhibitor, and the migration of AGS cells was detected by Transwell assay. (h) AGS cells were co-cultured with CAFs-EVs after transfection with miR-199a-5p inhibitor, and the invasion of AGS cells was detected by Transwell assay. (i) AGS cells were co-cultured with CAFs-EVs after transfected with miR-199a-5p inhibitor, and the effect of AGS cells on microtubule formation of HUVECs was detected by microtubule formation experiment. *p < 0.05. The experiment was repeated three times independently.

Figure 2.

miR-199a-5p inhibits FKBP5 and upregulates AKT1 phosphorylation. (a) Bioinformatics website (http://www.microrna.org/) was used to predict the binding site of miR-199a-5p with 3ʹUTR of FKBP5. (b) GEPIA database was used to analyze FKBP5 expression in gastric cancer. (c) Dual luciferase reporter gene assay was performed to verify the targeted binding of miR-199a-5p with FKBP5. (d) KEGG enrichment analysis of FKBP5 related genes. (e) Determination of binding of FKBP5 and AKT1 by Co-IP. (f) After transfection with miR-199a-5p mimic or inhibitor, the phosphorylation of FKBP5 and AKT1 was detected by Western blot. *p < 0.05. The experiment was repeated three times independently.

Figure 3.

miR-199a-5p upregulates AKT1 phosphorylation by inhibiting FKBP5 to induce malignant phenotypes of gastric cancer cells. (a) AGS cells were transfected with miR-199a-5p-mimic, oe-FKBP5 or MK-2206, and the proliferation of AGS cells was detected by EdU staining (200 ×). (b) AGS cells were transfected with miR-199a-5p-mimic, oe-FKBP5 or MK-2206, and Transwell assay was used to detect the migration of AGS cells. (c) AGS cells were transfected with miR-199a-5p-mimic, oe-FKBP5 or MK-2206, and the invasion of AGS cells was detected by Transwell assay. (d) After AGS cells were transfected with miR-199a-5p-mimic, oe-FKBP5 or MK-2206, the effect of AGS cells on microtubule formation of HUVECs was detected by microtubule formation experiment. (e) After AGS cells were transfected with miR-199a-5p-mimic, oe-FKBP5 or MK-2206, cell cycle distribution was detected by flow cytometry. *p < 0.05. The experiment was repeated three times independently.

Figure 4.

miR-199a-5p activates the AKT1/mTORC1 signaling pathway by upregulating AKT1 phosphorylation. (a) The phosphorylation of mTOR in AGS cells treated with miR-199a-5p-mimic or MK-2206 was detected by Western blot. (b) AGS cells were transfected with miR-199a-5p-mimic or temsirolimus, and their proliferation was detected by EdU staining. (c) AGS cells were transfected with miR-199a-5p-mimic or temsirolimus, and their migration was detected by Transwell assay. (d) After AGS cells were transfected with miR-199a-5p-mimic or temsirolimus, the invasion was detected by Transwell assay. (e) After AGS cells were transfected with miR-199a-5p-mimic or temsirolimus, the effect of AGS cells on microtubule formation of HUVECs was detected by microtubule formation experiment. *p < 0.05. The experiment was repeated three times independently.

Figure 5.

CAFs-EVs transfer miR-199a-5p to promote the tumorigenicity of gastric cancer cells in nude mice. (a) The tumor weight of nude mice injected with treated AGS cells. (b) Volume of the subcutaneous tumor in nude mice injected with treated AGS cells. (c) Immunohistochemical detection of Ki67 protein expression in the subcutaneous tumor tissues in nude mice injected with treated AGS cells. (d) The MVD in the subcutaneous tumor tissues in nude mice injected with treated AGS cells. (e) RT-qPCR detection of the expression of miR-199a-5p in the subcutaneous tumor tissues in nude mice injected with treated AGS cells. (f) Immunohistochemical detection of FKBP5 protein expression in the subcutaneous tumor tissues in nude mice injected with treated AGS cells. (g) The protein expression of FKBP5 and the phosphorylation level of AKT1 and mTORC1 were detected by Western blot. *p < 0.05. n = 10. The experiment was repeated three times independently.

Figure 6.

The molecular mechanism of CAFs-EVs containing miR-199a-5p in gastric cancer via regulation of the AKT1/mTORC1 signaling pathway mediated by FKBP5. CAFs-derived EVs transfer miR-199a-5p into gastric cancer cells, which downregulate FKBP5 to activate the AKT1/mTORC1 signaling pathway, thereby promoting the proliferation, invasion, migration and angiogenesis of gastric cancer cells and ultimately leading to the occurrence and development of gastric cancer.