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American Journal of Cancer Research logoLink to American Journal of Cancer Research
. 2021 May 15;11(5):2124–2141.

Exosomal transfer of miR-429 confers chemoresistance in epithelial ovarian cancer

Taoqiong Li 1,2,*, Li Lin 1,2,*, Qin Liu 4,*, Wujiang Gao 1,2, Lu Chen 1,2, Chunli Sha 1,2, Qi Chen 2, Wenlin Xu 2, Yuefeng Li 3, Xiaolan Zhu 1,2,5
PMCID: PMC8167704  PMID: 34094673

Abstract

The development of multidrug resistance during chemotherapy is the main obstacle for epithelial ovarian cancer (EOC) treatment. Exosomal transfer of carcinogenic microRNAs (miRNAs) might strengthen chemoresistance in recipient cells. Here, we identified through microarray analysis higher miR-429 expression in multidrug-resistant SKOV3 cells and their secreted exosomes (SKOV3-EXO) than in sensitive A2780 cells and their secreted exosomes. SKOV3-derived exosomes were internalized by A2780 cells, which permitted the transfer of miR-429. Exosomal miR-429 enhanced the proliferation and drug resistance of A2780 cells by targeting calcium-sensing receptor (CASR)/STAT3 pathway in vitro and in vivo. In addition, NF-κB-p65 was predicted to bind to the miR-429 promoter region, and the inhibition of NF-κB reduced the expression of miR-429 and led to the sensitivity of EOC cells. Consistently, A2780 cells co-incubated with SKOV3 pretreated with an NF-κB inhibitor or miR-429 antagomir showed sensitivity to cisplatin and exhibited attenuated cell proliferation. Based on our data, exosomal miR-429 functions as a primary regulator of the chemoresistance and malignant phenotypes of EOC by targeting CASR through a mechanism promoted by NF-κB and might be a therapeutic target for EOC.

Keywords: Exosome, miR-429, chemoresistance, NF-κB, EOC

Introduction

Epithelial ovarian cancer (EOC) accounts for the vast majority of ovarian cancers (OCs) [1]. The emergence of drug resistance during chemotherapy is an important cause of treatment failure in patients with EOC, and the mechanism has not yet been elucidated [2,3]. However, the acquisition of drug resistance not only occurs due to changes in the biological characteristics of the cell itself or repeated drug stimulation, as well as exogenous acceptance [4].

Exosomes are nano-scale microvesicles (30-100 nm in diameter) formed by the invagination of late endosomes released into the extracellular environment after fusion with the cell membrane [5,6]. The lipid membrane facilitates the uptake of exosomes by contiguous alternately inaccessible beneficiary cells; then, after uptake, the substances in exosomes exert their biological functions, which are known as the third cell division correspondence method [7]. The cells that take up exosomes, including malignant cells, might obtain RNA cargo, particularly exosomal microRNAs (miRNAs), which have attracted considerable attention from researchers. Exosomal miRNAs are protected from digestion by RNases because of the lipid membranes. New evidence has described important roles for exosomal miRNAs (EXO-miRNAs) secreted by tumor cells in regulating tumor growth, angiogenesis, metastasis, and chemotherapy resistance [7-10]. Interestingly, recent studies have shown that drug-resistant cancer cells develop chemoresistance to recipient cells following EXO-miRNA transformation. For example, doxorubicin chemoresistance is conferred by the exosomal delivery of miR-501 in gastric cancer [11], and exosomal delivery of microRNA-32-5p leads to drug resistance in hepatocellular carcinoma [12]. Furthermore, exosomes secreted from resistant cells confer characteristics of the malignant phenotype through the transfer of miRNA-222-3p in non-small cell lung cancer (NSCLC) [13].

This study aimed to explore whether miRNAs are transferred via exosomes and the effects of these exosomal miRNAs on drug resistance in EOC cells in vitro and in vivo. We also aimed to investigate the underlying mechanisms and potential prognostic biomarkers for EOC.

Materials and methods

Patients

3 EOC patients treated in our department from January 2021 to February 2021 were included in this study. These patients provided serum samples prior to chemotherapy. Three serum samples from normal people (healthy volunteers) were also collected as a negative control group. All serum samples were stored at -80°C for future research. This study was carried out in accordance with the 1975 Declaration of Helsinki. The Research Ethics Committee of the Fourth Affiliated Hospital of Jiangsu University approved the study. Each patient’s and healthy volunteer’s identification number was encrypted to protect their privacy; thus, the need for informed consent was waived. The characteristics of NC and EOC patients were provided in Table S1.

Cell culture

The human EOC cell lines A2780 and SKOV3 were purchased from iCell Bioscience Inc. (Shanghai, China). The A2780 and SKOV3 cell lines were authenticated by the short tandem repeat assay (STR). The cell lines were cultured in RPMI 1640 medium containing 10% FBS (Thermo Fisher, Carlsbad, CA). For the coculture experiment, A2780 cells were grown in the upper chamber of a 0.4-µm-pore size Transwell insert and SKOV3 cells were plated in the lower chamber of the Transwell chamber. All cells were cultured in medium supplemented with 10% exosome-free FBS at 37°C in the presence of 5% CO2.

Exosome isolation, quantification and labeling of cells

SKOV3 cells (1×106) were cultured in medium containing 10% exosome-free FBS for 48 h. Next, 40 ml of the conditioned medium was collected, and the collected supernatant was centrifuged at increasing speeds of 300×g for 10 min, 2000×g for 10 min, and 10,000×g at 4°C for 30 min to remove sediment and debris, thus obtaining pure exosomes. The supernatant was centrifuged at 100,000×g for 2 h to pellet the exosomes in clear ultracentrifuge tubes (Beckman Coulter, Indianapolis, USA). Transmission electron microscopy (TEM) was performed to verify the presence of exosomes. The exosomes were dissolved in PBS buffer, dropped onto a carbon-coated copper grid, and then stained with 2% uranyl acetate. The exosome images were obtained using an HT 7Tecnai G2 Spirit electron microscope (FEI Co, Oregon, USA). The exosomes were isolated from 40 ml of conditioned medium from SKOV3 cells by ultracentrifugation and then were resuspended in 100 µl of PBS. Before NTA, the SKOV3 cell-secreted exosomes were diluted by a factor of 100 with PBS to obtain an approximate number of vesicles of no more than 1×107. The size and concentration of the exosomes were analyzed using the ZetaView PMX 110 system (Particle Metrix, Meerbusch, Germany) and corresponding software (ZetaView 8.02.28). The exosomes from SKOV3 cells were labeled with PKH67 (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. The uptake of the labeled exosomes by A2780 cells was detected using a Leica TCS SP5 II laser scanning confocal microscope. GW4869 (Sigma-Aldrich, MO, USA) was utilized to block exosome formation and release at a concentration of 2.5 μM.

Exosomes isolation from patients serum

Frozen serum samples thawed in a 25°C water bath, completely thawed and placed on ice. Transfer serum to new tubes and centrifuged at 3000 g for 10 min at 4°C to remove cell debris. Transfer the supernatant to new tubes and centrifuged at 10,000 g for another 10 min at 4°C to remove impurities in the serum sample. Transfer the supernatant to new tubes and diluted 1:3 with pre-cooled PBS. Then add the same volume of Blood PureExo Solution (BPS, Umibio, Shanghai, China) as the serum sample, and vibrated for 1 min. Stood for 2 hours at 4°C. Centrifuged at 10000 g for 60 min at 4°C to obtain the sediment riched exosomal particles, stored at -80°C for subsequent studies.

Transfection

SKOV3 cells and A2780 cells (3×105/well) were respectively transfected with mimic, inhibitor and the corresponding negative control (mimics-NC, inhibitor-NC) (GenePharma, Shanghai, China) of miR-429, miR-200b-5p and miR-1269b at 100 nM using Lipofectamine 2000.Cotransfection of 50 nM mir-429 inhibitor and 50 nM small siRNA specific for CASR (GenePharma, Shanghai, China) was also included.

RNA extraction and real-time quantitative RT-PCR (Qrt-PCR) analysis

Total RNA was extracted from exosomes, SKOV3 cells, A2780 cells, cocultured A2780 cells (CO-A2780), and frozen xenograft tumor tissues using TRIzol reagent (Invitrogen, CA, USA). qRT-PCR analysis for miR-429, miR-200b-5p and miR-1269b and CASR mRNA was performed according to the manufacturer’s instructions. The relative expression of miR-429 was normalized to that of U6, and CASR mRNA expression was normalized to GAPDH; both were calculated using the 2 (-∆∆CT) method.

Western blot analysis

Proteins from cells, xenograft tumors and serum exosomes were lysed in equal volumes of ice-cold lysis buffer and a protease inhibitor cocktail. The cell and xenograft tumor lysates were separated by SDS-PAGE and then transferred to a 0.2 μm PVDF membrane (Bio-Rad, USA). After blocking, the membranes were immunoblotted with CASR, stat3, p-stat3, NF-κB, (1:1000; Abcam, Cambridge, MA) primary antibodies and then with horseradish peroxidase-conjugated secondary antibodies (dilution 1:5000). GAPDH (1:5000; Abcam, Cambridge, MA) was used as an endogenous control. The Odyssey Infrared Imaging System was used to visualize the targeted protein bands.

Apoptosis assay

Flow cytometry analysis was performed using the Annexin V-APC/7AAD Apoptosis Detection Kit (KeyGen BioTech, Jiangsu, China) according to the manufacturer’s instructions. Briefly, the cells were washed twice with cold PBS and then resuspended in 100 μl of 1× Binding Buffer, followed by the addition of 5 μl of Annexin V-APC and 5 μl of 7AAD and incubation for 15 min at room temperature in the dark. Thereafter, the cells were incubated with 400 μl of 1× Binding Buffer and analyzed by FACS Canto II flow cytometry (BD Biosciences).

RIP assay

The RIP assay was conducted using the RiboCluster Profiler RIP Assay Kit (MBL Medical & Biological Laboratories Co., Ltd., Japan) according to the manufacturer’s instructions. The cells (1×107) were lysed in RIP lysis buffer containing protease inhibitors and an RNase inhibitor. Protein A/G magnetic beads were incubated with a CASR antibody (Abcam) or NC rabbit IgG overnight at 4°C. The precipitated RNAs were separated with TRIzol reagent, reverse transcribed using the Revert Aid Kit (Thermo) and subsequently detected with qPCR. All the samples were detected in triplicate using independent tests.

Mouse xenograft tumor model

Thirty 6-week-old female BALB/c nude mice were purchased from the Shanghai SLAC Laboratory Animal Co, Ltd. (Shanghai, China) and housed under specific pathogen-free (SPF) conditions. All animal experiments were conducted according to the guidelines of the Medical Laboratory Animal Health Committee of Jiangsu University. Mice were injected subcutaneously with A2780 cells (1.0×107) and randomly divided into 5 groups (n = 6 mice per group). We measured the volumes of the subcutaneous tumors in nude mice once every 2 days. SKOV3 cells were transfected with the miR-429 agomir or antagomir. When the volume of the xenografts reached approximately 50 mm3, SKOV3 exosomes (50 μg) or PBS were intratumorally injected into the xenograft tumors three times a week for one week. All groups were then administered cisplatin (DDP, 5 mg/kg) three times a week for two weeks. On day 35, the mice in all groups were sacrificed, and tumors were harvested and frozen in liquid nitrogen.

Immunohistochemistry

Xenograft tumors were fixed in 4% PFA and embedded in paraffin wax. The tumor tissues were sectioned at 4-μm thickness, dewaxed in gradient alcohol and xylene, and incubated with the primary antibodies KI-67 and CASR (both from Abcam, Cambridge, UK) for 12 h at 4°C in a humid environment. The sections were then incubated with a biotinylated secondary antibody for 1 h at 37°C.

ChIP assay

Chromatin immunoprecipitation (ChIP) ChIP assays were conducted with the SimpleChIPTM ENZYMATIC Chromatin IP Kit (Magnetic Beads, #90003, CST, US) according to the manufacturer’s guidelines. Immunoselection of cross-linked protein-DNA was performed using the anti-NF-kB-P65 antibody and magnetic beads at 4°C overnight. Anti-rabbit IgG was used as a negative control. The purified DNAs were analyzed using qRT-PCR. The forward and reverse primers sequences of p65-chip-343, p65-chip-283, p65-chip-242 for the hsa-miR-429 promoter were provided in Table S7.

Statistical analysis

Statistical analyses were conducted using SPSS software. At least three biological replicates were performed in each experiment in our study, and the data are presented as the means ± SD. The statistical significance of differences between two groups was assessed using two-tailed Student’s t test. One-way ANOVA was adopted to compare the means among multiple groups. *P < 0.05, **P < 0.01, AND ***P < 0.001.

Results

The cisplatin (DDP) resistance of A2780 cells is enhanced after coculture with SKOV3 cells

The half-maximal inhibitory concentrations (IC50s) of cisplatin (DDP), paclitaxel (PTX) and cytoxan (CTX) were increased in SKOV3 cells compared with A2780 cells (Figure 1A), indicating that A2780 cells are more sensitive to DDP, PTX and CTX than SKOV3 cells. A2780 cells were cocultured with SKOV3 cells to determine the effects of SKOV3 cells on the DDP resistance of A2780 cells (Figure 1B). As expected, after coculture with SKOV3 cells, A2780 cells exhibited increased cell viability, enhanced drug resistance and a reduced apoptosis rate (Figure 1C-F). Based on these data, the drug resistance of A2780 cells was enhanced by SKOV3 cells.

Figure 1.

Figure 1

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Drug resistance identification and the resistance effect of SKOV3 cells on A2780 cells. (A) IC50 for DDP, PTX and CTX in SKOV3 cells and A2780 cells. (B) Schematic diagram of coculture. Cell viability (C) and IC50s for DDP (D) of A2780 cells and A2780 cells cocultured with SKOV3 cells. Apoptosis (E) and the related quantitative analysis of apoptosis (F) of A2780 cells and A2780 cells cocultured with SKOV3 cells.

SKOV3-secreted exosomes confer chemoresistance to recipient A2780 cells

We isolate exosomes from the conditioned medium of SKOV3 cells to explore the roles of exosomal delivery in DDP resistance. The double-layered vesicles displayed a round shape with a diameter ranging from 50 nm to 200 nm under TEM (Figure 2A). The Nanosight particle tracking analysis (NTA) further confirmed that the size of most vesicles was 80 nm after multiple dilutions (Figure 2B). The exosomal markers CD63 and CD81 were detected in the exosomes using a flow cytometry analysis (Figure 2C). Thus, the vesicles secreted from SKOV3 cells display typical characteristics of exosomes.

Figure 2.

Figure 2

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SKOV3 cells enhance the chemoresistance of A2780 recipient cell exosomes. (A) Transmission electron micrography showed round-shaped vesicles with bilayered membranes ranging from 50 nm to 200 nm in diameter released by SKOV3 (SKOV3-EXO). Scale bar: 100 nm. (B) Nanosight particle tracking analysis (NTA) indicated that the dominant size of SKOV3-EXO was approximately 100 nm. (C) FCM analysis of exosomal marker proteins CD63 and CD81 in SKOV3-EXO. (D) Schematic diagram of A2780 cells cocultured with SKOV3-EXO. (E) Confocal microscopy showed exosome internalization by A2780 recipient cells after coincubation with PKH67-labeled (green fluorescence) SKOV3-EXO. DAPI was used to stain the nuclei of A2780 recipient cells with blue fluorescence. Scale bar = 10 µm. Cell viability (#: PBS vs SKOV3-EXO, *: CO-culture+DMSO vs CO-culture+GW4869) (F) and IC50s for DDP (G) of cocultured A2780+DMSO, cocultured A2780+GW4869, A2780+PBS, and A2780+SKOV3-EXO.

Next, we used the fluorescent tracer PKH67 to label SKOV3-EXO and visualize exosomal delivery. After incubating the labeled SKOV3-EXO with A2780 cells, strong green fluorescence was observed in the cytoplasm of the recipient cells (A2780 cells) with a confocal microscope (Figure 2D, 2E), suggesting that the recipient A2780 cells successfully absorbed the exosomes derived from SKOV3 cells.

GW4869 was employed to prevent exosome secretion from SKOV3 cells plated in the lower chamber of a coculture system with 0.4-µm pores (Corning. American), and A2780 cells were plated in the upper chamber and collected for subsequent experiments to further determine whether the transfer of drug resistance from SKOV3 cells to A2780 cells depend on exosomes (Figure 1B). The viability and DDP resistance of A2780 cells were decreased upon coculture with SKOV3 cells pretreated with GW4869. Next, SKOV3-EXO was directly added to the medium of A2780 cells (A2780 + SKOV3-EXO). The CCK8 assay showed an increase in the viability of A2780 cells treated with SKOV3-EXO. The IC50 of DDP was increased in A2780 cells incubated with SKOV3-EXO compared with A2780 cells cocultured with an equal amount of PBS (Figure 2F, 2G). In summary, these results revealed that exosomes secreted from the drug-resistant EOC cells were transferred to the adjacent drug-sensitive EOC cells, thus conferring chemoresistance to DDP.

Identification and screening of differentially expressed miRNAs in A2780 cells compared with SKOV3 cells and A2780-EXO compared with SKOV3-EXO

We detected the expression profile of microRNAs in A2780 compared with SKOV3 cells and A2780-EXO compared with SKOV3-EXO using next-generation sequencing (NGS) (Guangzhou RiboBio Co., Ltd.) to evaluate the role of exosomal miRNAs in the drug resistance mechanism of EOC cells. The original sequencing data was provided in the supplementary material. Significant differentially expressed miRNAs were selected based on log2 (fold change) ≥10. The heatmap showed higher expression of 16 miRNAs and lower expression of 17 miRNAs in SKOV3 cells than in A2780 cells (Figure 3A, left panel). The heatmap also showed higher expression of 15 miRNAs and lower expression of 2 miRNAs in SKOV3-EXO than in A2780-EXO (Figure 3A, right panel). Next, the Venn diagram revealed three miRNAs (miR-429, miR-200b-5p and miR-1269b) with higher expression in both comparison groups (Figure 3B). We used RT-PCR to verify the expression of the upregulated miRNAs mentioned above. Nine miRNAs were expressed at higher levels in SKOV3 cells than in A2780 cells, and 5 miRNAs were expressed at higher levels in SKOV3-EXO than in A2780-EXO. Three miRNAs (miR-429, miR-200b-5p and miR-1269b) displayed higher expression in both comparison groups, consistent with the NGS results. (The results of the PCR verification of the three miRNAs are shown in Figure 3C; the results of the verification of other miRNAs are presented in Figure S1). Based on these results, three miRNAs may serve as potential messengers of drug resistance information and play important roles in the drug resistance of EOC. The three miR-RNAs had higher expression in SKOV3 than A2780 in both cell and exosomes. The expression difference of miR-429 in SKOV3-EXO and A2780-EXO was the most obvious. And in clinical samples, the expression of miR-429 in exosomes from peripheral blood of ovarian cancer also increased significantly, compared to normal people Figure S2.

Figure 3.

Figure 3

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Identification and screening of miRNAs in A2780 cells vs SKOV3 cells and A2780-EXO vs SKOV3-EXO. A. The heatmap showed that 16 miRNAs have higher expression levels and 17 miRNAs have lower expression levels in SKOV3 cells than in A2780 cells (left panel); 15 miRNAs have higher expression levels and 2 miRNAs have lower expression levels in SKOV3-EXO compared with those in A2780-EXO (right panel). B. The Venn diagram shows three miRNAs (miR-429, miR-200b-5p and miR-1269b) with higher expression in both comparison groups. C. Real-time qRT-PCR revealed that the levels of miR-429, miR-200b-5p and miR-1269b were higher in SKOV3 cells than in A2780 cells and that the levels were higher in SKOV3-EXO than in A2780-EXO.

The effect of miR-429 on the chemoresistance of A2780 cells is mediated by targeting CASR

According to the NGS results, three miRNAs (miR-429, miR-200b-5p and miR-1269b) were highly related to chemoresistance in EOC cells. A2780 cells (3×105/well) were transfected with the agomirs of miR-429, miR-200b-5p or miR-1269b or with agomir-NC at 100 nM to explore the functions of these miRNAs in drug resistance. A2780 cells transfected with miR-429-agomir showed a higher IC50 than A2780 cells transfected with miR-200b-5p-agomir or miR-1269b-agomir (Figure 4A). Hence, miR-429 had the strongest contribution to drug resistance. Thereafter, we verified whether miR-429 was delivered by exosomes. SKOV3 cells transfected with Cy3-miR-429 mimic were plated in the lower chamber of the coculture system. After 48 hours of cocultivation, the A2780 cells inoculated in the upper chamber displayed strong red fluorescence (Figure 4B). Therefore, miR-429 may be delivered from SKOV3 cells to A2780 cells by exosomes. A2780 cells (3×105/well) were transfected with the agomir, antagomir or the negative control (agomir-NC or antagomir-NC) of miR-429 at 100 nM (chosen for transfected cell lines substantiated by qRT-PCR, Figure S3A) to further determine the effect of miR-429 on the chemoresistance of A2780 cells. A2780 cells transfected with the agomir showed increased drug resistance, and A2780 cells transfected with the antagomir were much more sensitive to DDP (Figure 4C). These results indicate that miR-429 overexpression contributed to drug resistance in vitro.

Figure 4.

Figure 4

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Chemoresistant effect of miR-429 on A2780 cells by CASR/STAT3 pathway. (A) After transfection with the agomirs of miR-429, miR-200b-5p and miR-1269b, the A2780 cells were treated with different concentrations of DDP for 48 h. DDP resistance was significantly enhanced in A2780 miR-429 agomir cells compared with that in A2780 miR-200b-5p agomir cells and A2780 miR-1269b agomir cells. (B) SKOV3 cells transfected with the Cy3-miR-429 mimic (red fluorescence) were plated in the lower chamber and coincubated with A2780 cells seeded in the upper chamber in a coculture system with a 0.4-µm pore membrane. Red fluorescence was observed in the A2780 recipient cells under the fluorescence microscope. Scale bar = 10 µm. IC50 for DDP (C), fold change of CASR mRNA (D) and CASR, STAT3, p-STAT3 protein levels (E) in A2780 miR-429 agomir, A2780 miR-429 agomir NC, A2780 miR-429 antagomir, and A2780 miR-429 antagomir NC. (F) RIP assays using an anti-CASR antibody showed that CASR interacts with miR-429 in A2780 cells, followed by qRT-PCR analysis of miR-429 in immunoprecipitated RNAs. The CASR, STAT3, p-STAT3 protein levels (G) (The original uncropped gels of Western blot assay were shown in Figure S6) and IC50s for DDP (H) of A2780 miR-429 antagomir NC, A2780 miR-429 antagomir, A2780 miR-429 antagomir+siCASR-NC, A2780 miR-429 antagomir+siCASR.

Notably, miRNAs to the 3’-untranslated region (3’-UTR) of the target mRNA to directly degrade the mRNA or inhibit post-transcriptional protein translation and negatively regulate gene expression [14-16]. The selected target genes of miR-429 were predicted by TargetScan, miRanda, and miRWalk. Since these programs have a certain false positive rate, only the genes predicted by all three programs were proposed to be miRNA target genes. One hundred eighty-six genes were predicted to be targeted by miR-429 (Table S2). One miRNA may regulate the expression of multiple target genes, and the one gene might also be targeted by several miRNAs [17,18]. According to the results of target gene prediction, calcium-sensing receptor (CASR) showed the highest align_score in miRanda, 4 binding sites in CASR were predicted by miRWalk, all with binding probabilities of 100%, and the site type of CASR was 8mer-1a in TargetScan (Figure S3B; Tables S3, S4, S5). In summary, CASR was identified by the three target gene prediction websites and showed a good score. Additionally, CASR was confirmed to be associated with chemotherapy resistance in colon cancer and breast cancer [19-21]. After considering these findings, we chose CASR as the target gene for subsequent study. We detected the changes in the CASR mRNA and protein levels in A2780 cells transfected with the agomir and antagomir to assess the effect of miR-429 on CASR expression. The levels of the CASR mRNA and the proteins expression of CASR were attenuated in the agomir group and elevated in the antagomir group; the expression of stat3 and p-stat3 were elevated in the agomir group and attenuated in the antagomir group (Figure 4D, 4E). RIP assays were conducted in A2780 cells using the IgG antibody, followed by qRT-PCR analysis of miR-429 in immunoprecipitated RNAs to further confirm the relationship between CASR and miR-429. The RIP results indicated that miR-429 was enriched in the RNA immunoprecipitate using the anti-CASR antibody compared to IgG (Figure 4F), supporting the hypothesis that CASR interacts with miR-429 in A2780 cells. Then, we inhibited CASR expression on the basis of the miR-429 antagomir transfection. The inhibition of CASR could largely rescue the increase in CASR expression level and the decrease in drug resistance via stat3 pathway caused by miR-429 antagomir (Figure 4G, 4H) (The infection efficiency of si-CASR was confirmed by Western blot analyses in Figure S3D, the original uncropped gels of Western blot assay was shown in Figure S7). These data further verified that miR-429 conferred DDP resistance to A2780 cells via CASR/ STAT3 pathway.

Exosomal miR-429 induces drug resistance in EOC in vivo via CASR/STAT3 pathway

We subcutaneously injected A2780 cells into female mice to produce appropriately sized tumors and study the function of miR-429 present in SKOV3-EXO in tumor growth and chemoresistance. Exosomes (50 μg) (Figure S4) or PBS were injected into the xenograft tumors three times a week for one week (n = 6). We provide a simple flow chart of the animal experiments in Figure 5A. Xenografts injected with miR-429-overexpressing SKOV3-EXO grew significantly larger. In contrast, miR-429 inhibition abrogated SKOV3-EXO-regulated tumor growth in mice (Figure 5B, 5C). We quantified the expression of CASR and a tumor cell proliferation marker (KI67). Xenografts injected with miR-429-overexpressing SKOV3-EXO displayed a remarkable increase in the numbers of KI67-positive (KI67+) cells and decreased CASR expression (Figure 5D). Xenografts injected with miR-429-overexpressing SKOV3-EXO showed higher miR-429 expression, lower CASR expression and higher p- STAT3 expression (Figure 5E, 5F). A reduced number of KI67 + cells, a decrease in miR-429 expression, an increase in CASR expression and a decrease p- STAT3 expression were observed in xenografts treated with exosomes containing the miR-429 inhibitor. These results further support the hypothesis that the exosomal transfer of miR-429 downregulated CASR and impaired the therapeutic effects of DDP in vivo.

Figure 5.

Figure 5

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Effect of exosomal miR-429 in DDP resistance in vivo. Exosomes derived from SKOV3 cells were transfected with agomir or antagomir (SKOV3-EXO-agomir-NC, SKOV3-EXO-agomir, SKOV3-EXO-antagomir-NC, and SKOV3-EXO-antagomir). A2780 cells were subcutaneously injected into BALB/c nude mice, and potential tumors were allowed to grow for a week. Next, exosomes (50 μg) or PBS was intratumorally injected into the xenograft tumors three times per week for one week (n = 6). All the groups were administered DDP (5 mg/kg) by intraperitoneal injection three times a week for 2 weeks and were sacrificed. A. Flow chart of the animal experiment. B. Representative images of the excised tumors on day 35 after tumor cell injection. C. Tumor volumes of the excised tumors on day 35 after tumor cell injection. D. Immunohistochemistry analyses for KI-67 and CASR staining were carried out on A2780 xenograft tumor sections. Representative staining is shown (×200 magnification). E. The expression of miR-429 was higher in A2780+SKOV3-EXO-agomir cells than in A2780+PBS cells. The expression of miR-429 was lower in A2780+SKOV3-EXO-antagomir cells than in A2780+PBS cells. F. The level of CASR, STAT3, p-STAT3 proteins were downregulated in the A2780+SKOV3-EXO-agomir group, and the level of CASR protein was upregulated in the A2780+SKOV3-EXO-antagomir group, as assessed by Western blotting in mouse xenograft tumor tissues. The original uncropped gels of Western blot assay was shown in Figure S6. *P < 0.05 and **P < 0.01. ***P < 0.001.

Exosomal miR-429 promoted by NF-κB functions as a principal regulator of drug resistance and the malignant characteristics of EOC

Because increased miR-429 expression caused drug resistance, we sought to identify factors that might affect miR-429 expression using a transcription factor prediction website. Many transcription factors were involved in the transcription of miR-429. We predicted the transcription factors regulating miR-429 using the TRANSFAC 8.3 database according to the sequence of the miR-429 promoter region (2000 bp upstream and 1 bp upstream). We have provided the transcription factor prediction results for miR-429 in Table S6. According to the prediction, we found that there were three NF-κB binding sites in the promoter region of miR-429 with high specificity. NF-κB was associated with the resistance of various tumors. Therefore, we predicted that NF-κB might be the transcription factor regulating miR-429. Then we detected the effect of NF-κB on miR-429 expression. We treated SKOV3 and A2780 cells with gradient concentrations of the NF-κB inhibitor PDTC. Notably, miR-429 was downregulated in SKOV3 and A2780 cells treated with increased concentrations of PDTC, according to our tests (Figure 6A, 6B). Furthermore, we conducted experiments to explore the effect of NF-κB on the drug resistance of EOC. After treatment with 200 μM PDTC, the viability and IC50 of SKOV3 and A2780 cells were both decreased (Figure 6C, 6D). Based on these results, NF-κB promoted drug resistance in ovarian cancer (OC). Next, we cocultured A2780 cells with PDTC-treated SKOV3 cells to determine whether the regulatory effect of NF-κB on EOC resistance was related to miR-429. The viability and IC50 of A2780 cells decreased compared with A2780 cells cocultured with SKOV3 cells (Figure 6E). The viability and IC50 of A2780 cells were also attenuated upon coculture with SKOV3-antagomir-treated SKOV3 cells (Figure 6F). These data further illustrate that NF-κB promotes drug resistance and may be related to miR-429 expression. According to the results of the ChIP-qPCR assay in SKOV3 and A2780 cells, p65-chip-343, p65-chip-283, p65-chip-242 could bound to miR-429 promoter and the enrichment efficiency of p65-chip-343 was more than 10 fold changes (Figure 6G).

Figure 6.

Figure 6

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NF-κB promotes the transcription of miR-429 to mediate drug resistance. After treatment with different concentrations of PDTC for 24 h, NF-κB p65 protein expression in SKOV3 and A2780 cells were measured by Western blotting (A). The original uncropped gels of Western blot assay were shown in Figure S7. Additionally, the expression of miR-429 was detected by real-time qRT-PCR (B). Cell viability and IC50s for DDP of SKOV3 cells (C) and A2780 cells (D) treated with 0 or 200 μM PDTC (E). Cell viability and IC50s for DDP of A2780 cells cocultured with SKOV3 cells treated with 0 or 200 μM PDTC (F). Cell viability and IC50s for DDP of A2780 cells cocultured with SKOV3 transfected with miR-429 antagomir. The chrome immunoprecipitations (CHIP) were performed by using specific anti-NF-κB-p65 (G).

Based on these results, we proposed a model for the underlying effects of exosomal miR-429 on EOC (the schematic diagram was presented in Figure S5). SKOV3 cell-derived exosomes containing miR-429 were taken up by the recipient cells and enhanced drug resistance. NF-κB may increase drug resistance by promoting miR-429 transcription.

Discussion

Platinum-based therapy is the main treatment option for OC, while chemoresistance often leads to therapeutic failure [22,23]. Exosomes have been reported to play an important role in chemoresistance as novel intracellular communication molecules [24-26]. Recently, miRNAs were observed to be loaded in exosomes to escape degradation [27]. These exosomal miRNAs are shuttled into the recipient cells and change the phenotype of these cells by promoting changes in gene expression [28,29]. Chemotherapy-resistant tumor cells may release exosomal miRNAs to the microenvironment and endow the recipient cells with drug resistance [30-32]. As shown in the study by Wang et al., the exosomal transfer of miR-155-5p from the paclitaxel-resistant MGC-803 gastric cancer cells promoted the EMT and drug resistance in paclitaxel-sensitive cells [33]. Exosomes decrease the adriamycin sensitivity of breast cancer cells by transferring miRNAs [8].

We identified the differentially expressed miRNAs in drug-resistant and drug-sensitive cells and their secreted exosomes by performing a microarray analysis to detect the messengers of drug resistance information in OC. Significant differentially expressed miRNAs were selected based on log2 (fold change) ≥10. After qRT-PCR identification, we discovered 9 miRNAs with higher expression levels in SKOV3 cells than in A2780 cells, and 5 miRNAs displayed higher expression levels in SKOV3-EXO than in A2780-EXO, as shown in the heatmap. Higher expression of miR-429 was detected in SKOV3 cells than in A2780 cells, and miR-429 was expressed at significantly higher levels in SKOV3-EXO than in A2780-EXO. SKOV3-EXO were taken up by recipient cells and maintained their effects, suggesting that miR-429 may be appropriate for packaging into exosomes to retain its stability and subsequently play a significant role in chemoresistance. In our study, high expression of miR-429 increased cell viability and drug resistance. Based on accumulating evidence, miR-429 dysregulation is related to the EMT, progression, development, invasion, metastasis, apoptosis and drug resistance of various cancers [34]. Combined with the results of NGS and assessments of cellular function, we selected miR-429 as the candidate miRNA. Notably, miR-429 belongs to the miR-200 family, which includes miR-200a, miR-200b, miR-200c, miR-141 and miR-429 and is located on chromosome 1 [35]. Previous studies have reported negative correlations between the miR-429 expression level and renal cell carcinoma (RCC) [36], breast cancer (BC) [37], gastric carcinoma (GC) [38], glioblastoma (GBM) [39], esophageal cancer (EC) [40], osteosarcoma [41], and cervical cancer (CC) [42], and miR-429 is a negative indicator of the drug resistance of these cancer types. The potential explanation is that miR-429 is relevant to tumorigenesis in a specific pattern, which may function as a tumor suppressor or promote the expression of certain cancer candidate genes in specific types of tumor cells/tissues. Moreover, the ability of miR-429 to enhance drug resistance was also consistent with previous research showing that miR-429 increased endometrial carcinoma, prostate cancer and lung cancer cell resistance. However, However, the functions of exosomal miR-429 in the chemoresistance of EOC remain to be elucidated [35].

In the present study, exosomal miR-429 released from SKOV3 cells was internalized by recipient cells. Exosomal miR-429 uptake increased the DDP resistance of recipient cells in vitro and in vivo, and exosomal miR-429 may function by targeting CASR. According to the GEPIA database (Figure S3C), the expression of CASR in OC was higher than in normal tissues. CASR was a class-CG protein-coupled receptor that played a key role in the process of calcium transformation, mainly regulating the secretion of parathyroid hormone to maintain systemic calcium homeostasis [19]. CASR promoted and maintained the malignant and drug-resistant phenotype of colon cancer and was a robust promoter of colonic epithelial cell differentiation that functions as a tumor suppressor in colon cancer. CASR also exerted anti-tumor effects on breast cancer and promotes the sensitivity of cells to cytotoxic drugs [21]. Hence, the CASR gene may be a novel potential tumor suppressor. Through a series of analyses, including qRT-PCR, Western blotting and RIP, miR-429 was confirmed to suppress CASR expression in A2780 cells. Previous studies reported that CASR was involved in cardiomyocyte apoptosis through the activator of transcription 3 (STAT3) signaling pathway [43]. In addition, STAT3 proteins played important roles in cancer cell survival and proliferation [44,45]. Since CASR could activate the STAT3 pathway, and the activation of the STAT3 pathway promoted the development of drug resistance in ovarian cancer, we detected the STAT3 and p-STAT3 expression levels in vivo and in vitro experiments. We found that the expression level of p-STAT3 was negatively correlated with the expression level of CASR, which meant that CASR exerting as tumor suppressor may be achieved by inhibiting p-STAT3 in EOC cells, thereby promoting cell apoptosis and inhibiting cell proliferation. In this study, we verified that exosomal miR-429 secreted from SKOV3 cells enhanced DDP drug resistance in recipient cells via CASR/STAT3 in vivo.

According to the results of the transcription factor prediction analysis, we selected NF-κB for further research. NF-κB was the main transcription factor related to the induction of drug resistance. Moreover, in our experiments, the inhibition of NF-κB decreased the expression of miR-429 and the drug resistance of ovarian cancer cells. The NF-κB family consists of five related transcription factors: p50, p52, RelA (p65), c-Rel and RelB1,2. According to the predicted results, three possible binding sites for NF-κB were present in the miR-429 promoter region. NF-κB was a ubiquitous transcription factor that mediates cytoplasmic/nuclear signaling pathways [46]. A correlation has been identified between the activation of NF-κB and the control of apoptotic pathways, cell proliferation, differentiation, migration, angiogenesis, and resistance to chemotherapy/radiotherapy in tumor cells [47-50]. In particular, the overactivation of NF-κB led to resistance to standard chemotherapy agents, including PTX and DDP in OC [51,52]. The inhibition of NF-κB decreased the expression of miR-429 and led to the sensitivity of OC cells. Additionally, A2780 cells cocultured with SKOV3 cells before treatment with the NF-κB inhibitor (PDTC) or miR-429 antagomir were sensitive to DDP and exhibited attenuated cell proliferation. In terms of inhibiting drug resistance, the inhibitory effect of NF-κB was greater than the miR-429-antagomir. We postulated that NF-κB might participate in the transcriptional regulation of multiple miRNAs. NF-κB was very likely to have a transcriptional regulatory effect on miR-429. Above all, NF-κB was very likely to have a transcriptional regulatory effect on miR-429.To further confirm this, we conducted a CHIP assay. According to the predicted three binding sites, we designed three pairs of primers: p65-chip-343, p65-chip-283, p65-chip-242, among which the enrichment efficiency of p65-chip-343 was more than 10 fold changes in SKOV3 and A2780 cells. The CHIP results supported that NF-κB had a transcriptional regulation effect on miR-429. NF-κB had several subunits, while p65 was the core subunit of transcription regulation, and it was in the predicted three NF-κB binding sites. Among the three predicted sites, two binding sites were RelA (p65), in addition, the antibody we used in the CHIP experiment was NF-κB-p65, so we thought that NF-κB-p65 could bind to the miR-429 promoter region, and promote miR-429 transcription.

In our study, the mechanism of that NF-κB regulated miR-429 transcription and the identification of other downstream factors that conferred the drug resistance effect of miR-429 require further investigation. Overall, our current results indicated that DDP resistance was conferred by the horizontal transfer of exosomal miR-429 that subsequently inhibits CASR expression in EOC. Exosomal miR-429 might be a novel therapeutic target for EOC.

Acknowledgements

This research was supported by the Natural Science Foundation of China (Grant Nos. 81672913 and 81871343), the Social Development Project of Jiangsu (Grant Nos. BE2018693 and BE2017698), the Natural Science Foundation of Jiangsu Province (Grant No. BK20181226 and BK20201223), and Jiangsu Provincial Medical Youth Talent (Grant Nos. FRC201788 and QNRC2016460).

Disclosure of conflict of interest

None.

Supporting Information

ajcr0011-2124-f7.pdf (1.7MB, pdf)

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