Extracellular vesicle-encapsulated microRNA-296-3p from cancer-associated fibroblasts promotes ovarian cancer development through regulation of the PTEN/AKT and SOCS6/STAT3 pathways

Abstract

Cancer-associated fibroblasts (CAFs), as important components of the tumor microenvironment, can regulate intercellular communication and tumor development by secreting extracellular vesicles (EVs). However, the role of CAF-derived EVs in ovarian cancer has not been fully elucidated. Here, using an EV-microRNA sequencing analysis, we reveal specific overexpression of microRNA (miR)-296-3p in activated CAF-derived EVs, which can be transferred to tumor cells to regulate the malignant phenotypes of ovarian cancer cells. Moreover, overexpression of miR-296-3p significantly promotes the proliferation, migration, invasion, and drug resistance of ovarian cancer cells in vitro, as well as tumor growth in vivo, while its inhibition has the opposite effects. Further mechanistic studies reveal that miR-296-3p promotes ovarian cancer progression by directly targeting PTEN and SOCS6 and activating AKT and STAT3 signaling pathways. Importantly, increased expression of miR-296-3p encapsulated in plasma EVs is closely correlated with tumorigenesis and chemoresistance in patients with ovarian cancer. Our results highlight the cancer-promoting role of CAF-derived EVs carrying miR-296-3p in ovarian cancer progression for the first time, and suggest that miR-296-3p encapsulated in CAF-derived EVs could be a diagnostic biomarker and therapeutic target for ovarian cancer.

Keywords: cancer progression; cancer-associated fibroblast; extracellular vesicle; miR-296-3p; ovarian cancer.

FIGURE 1

Activated cancer‐associated fibroblast (CAF)‐derived extracellular vesicles EVs promote the proliferation, migration, and invasion of ovarian cancer cells. (A) Transmission electron microscopy analysis of EVs isolated from quiescent CAFs (qCAFs) and activated CAFs (aCAFs); the white arrowhead signifies EVs (scale bar, 500 nm). (B) Western blot analysis of CD63, TSG101, HSP70, and GM130 protein expression in EVs and cell lysates. (C, D) Uptake of PKH26‐labeled EVs by A2780 and SKOV3 cells was detected by immunofluorescence (scale bar, 20 μm). (E–G) A2780 and SKOV3 cells were treated with PBS, qCAF‐derived EV‐depleted conditioned medium (qCAF‐EV‐DP‐CM), qCAF‐derived EVs (qCAF‐EVs), aCAF‐derived EV‐depleted conditioned medium (aCAF‐EV‐DP‐CM), and aCAF‐derived EVs (aCAF‐EVs). (E) Cell proliferation, (F) migration, and (G) invasion abilities were investigated by EdU, Transwell migration, and invasion assays, respectively. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. ns, no significance.

FIGURE 2

MicroRNA (miR)‐296‐3p is specifically upregulated in activated cancer‐associated fibroblast (CAF)‐derived extracellular vesicles (EVs) from ovarian cancer. (A) Volcano plot showing the differentially expressed miRNAs between quiescent CAF (qCAF)‐ and activated CAF (aCAF)‐derived EVs. (B) Real‐time PCR analysis of miR‐296‐3p expression in qCAFs, aCAFs, and their respective EVs. (C) dbDEMC database analysis of miR‐296‐3p levels in normal ovarian tissues and ovarian cancer tissues. (D) Bioinformatics analysis of miR‐296‐3p expression in ovarian cancer based on tumor stages using the UALCAN database. (E) Real‐time PCR showing the expression of miR‐296‐3p in normal ovarian epithelial cells (HOSE 6‐3 and HOSE 17‐1), ovarian cancer cells (A2780, SKOV3, HO8910, OVCAR5, OV90, CAOV3, and ES‐2), normal fibroblasts (NFs), and CAFs. (F) CAF markers, including α‐smooth muscle actin (α‐SMA), vimentin, and FAP, were determined in three pairs of NFs and CAFs in colorectal cancer by real‐time PCR. (G) Real‐time PCR showing the expression of miR‐296‐3p in three pairs of NFs and CAFs in colorectal cancer. (H) EVmiRNA database analysis of miR‐296‐3p expression in EVs from different sample sources. Red arrows indicate the miR‐296‐3p expression in EVs from fibroblasts. (I) Real‐time PCR showing the expression of miR‐296‐3p in NFs, CAFs, and their respective EVs in ovarian cancer. (J) Relative expression of miR‐296‐3p in tumor cells after incubation with blank or qCAF‐ or aCAF‐derived EVs for 24 or 48 h. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. ns, no significance; RPM, reads per million; TCGA, The Cancer Genome Atlas.

FIGURE 3

MicroRNA (miR)‐296‐3p promotes ovarian cancer development in vitro and in vivo. (A, B) Effects of miR‐296‐3p on the proliferation of A2780 and SKOV3 cells were detected by EdU assays. (C, D) Cell colony‐forming ability was measured using colony formation assay. (E, F) Effects of miR‐296‐3p on the migration of A2780 and SKOV3 cells were determined with Transwell migration assays. (G, H) Representative images from Transwell invasion assays of A2780 and SKOV3 cells. (I, J) A2780 cells transfected with miR‐296‐3p or anti‐miR‐296‐3p and their negative control were used to establish s.c. xenograft tumors (n = 7). Tumor growth curves and tumor weights are shown. (K, L) Effects of miR‐296‐3p on the tumor growth of SKOV3 cells in nude mice (n = 7). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

FIGURE 4

MicroRNA (miR)‐296‐3p directly targets phosphatase and tensin homolog (PTEN) and suppressor of cytokine signaling 6 (SOCS6). (A) Schematic of the constructed luciferase reporter plasmid constructed containing WT and mutant (Mut) binding sites in the 3′‐UTR of PTEN and SOCS6 mRNA. (B) Analysis of dual‐luciferase reporter assays revealed the interaction between miR‐296‐3p with PTEN and SOCS6. (C) Relative mRNAs expressions of PTEN and SOCS6 were analyzed by real‐time PCR in A2780 and SKOV3 cells. (D) Western blot analysis of PTEN and SOCS6 protein levels in A2780 and SKOV3 cells. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. ns, no significance.

FIGURE 5

MicroRNA (miR)‐296‐3p mediates the proliferation, migration, and invasion of ovarian cancer cells in part through downregulation of phosphatase and tensin homolog (PTEN) and suppressor of cytokine signaling 6 (SOCS6). (A) Western blot analysis of PTEN and SOCS6 expression in A2780 and SKOV3 cells with PTEN or SOCS6 overexpression (OE). (B–E) Ectopic PTEN or SOCS6 expression in A2780 and SKOV3 cells. (B) Cell proliferation, (C) colony formation, (D) migration, and (E) invasion abilities were investigated by EdU, colony formation, Transwell migration, and invasion assays. (F) Western blot analysis of PTEN and SOCS6 levels in A2780 and SKOV3 cells cotransfected with miR‐296‐3p and PTEN or SOCS6. (G) Cell proliferation and (H) colony formation of A2780 and SKOV3 cells with PTEN or SOCS6 overexpression combined with or without miR‐296‐3p mimic. (I,J) Cell migration and invasion of A2780 and SKOV3 cells determined by Transwell assays. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. miR‐NC, mimic negative control.

FIGURE 6

MicroRNA (miR)‐296‐3p activates downstream AKT and signal transducer and activator of transcription 3 (STAT3) signaling pathways by targeting phosphatase and tensin homolog (PTEN) and suppressor of cytokine signaling 6 (SOCS6), respectively. (A, B) Western blot analysis of the critical mediators of AKT and STAT3 signaling pathways, including AKT, p‐AKT (S473), p‐AKT (T308), STAT3, and p‐STAT3 in A2780 and SKOV3 cells transfected miR‐296‐3p or anti‐miR‐296‐3p. (C) Overexpression (OE) of PTEN significantly reduced the p‐AKT (S473) and p‐AKT (T308) protein levels in A2780 and SKOV3 cells. (D) Relative protein expressions of AKT, p‐AKT (S473), and p‐AKT (T308) were analyzed by western blot in A2780 and SKOV3 cells cotransfected with PTEN and miR‐NC or miR‐296‐3p. (E) Western blot results demonstrate STAT3 and p‐STAT3 expression in A2780 and SKOV3 cells with SOCS6 overexpression. (F) Relative protein levels of STAT3 and p‐STAT3 were analyzed by western blot in A2780 and SKOV3 cells cotransfected with SOCS6 and miR‐NC or miR‐296‐3p. Band intensity was assessed with ImageJ. *p < 0.05; **p < 0.01; ***p < 0.001. ns, no significance. miR‐NC, mimic negative control.

FIGURE 7

MicroRNA (miR)‐296‐3p in plasma extracellular vesicles (EVs) is closely associated with tumorigenesis and chemoresistance in patients with ovarian cancer. (A) miR‐296‐3p expression in the blood circulation of patients with ovarian cancer and healthy controls was analyzed using the dbDEMC database. (B) EVs isolated from the plasma of patients with ovarian cancer and healthy controls were observed by transmission electron microscope; white arrowhead signifies EVs (scale bar, 500 nm). (C) Nanoparticle tracking analysis of the isolated EVs. (D) Western blot analysis of EV marker protein expression, including CD9, HSP70, Alix, and Annexin‐V. (E) The miR‐296‐3p expression in plasma EVs of patients with ovarian cancer and healthy controls was detected using real‐time PCR. (F) miR‐296‐3p expression in plasma EVs of drug‐sensitive and drug‐resistant patients with ovarian cancer. *p < 0.05; **p < 0.01. N‐EV, normal control‐derived EVs; T‐EV, tumor patien‐derived EVs.

FIGURE 8

Schematic diagram illustrating the regulatory axis of cancer‐associated fibroblast (CAF)‐derived extracellular vesicles (EVs) carrying microRNA (miR)‐296‐3p involved in promoting ovarian cancer development. PTEN, phosphatase and tensin homolog; SOCS6, suppressor of cytokine signaling 6; STAT3, signal transducer and activator of transcription 3.