Cancer-associated fibroblast-derived exosomal microRNA-98-5p promotes cisplatin resistance in ovarian cancer by targeting CDKN1A

In total, 42 patients with OC (aged 45–72 years; an average age of 53.52 ± 8.08 years old) who underwent surgical resection at the General Hospital of Ningxia Medical University from August 2017 to October 2018 were enrolled in this study. Based on the FIGO 2008 staging criteria, among the 42 patients, 26 patients were in stage I-II and 16 patients in stage III. Thirty normal ovarian tissues were collected as controls from healthy individuals (aged 42–71 years, with an average age of 54.33 ± 6.94 years old) who underwent ovariectomy during the same period. The OC tissues and normal ovarian tissues were fresh and intact and histopathologically confirmed. These tissue were rinsed with phosphate-buffer saline (PBS) containing 20% antibiotics, and then detached with collagenase I (Sigma-Aldrich Chemical Company, St Louis, MO, USA) and hyaluronidase (Sigma-Aldrich Chemical Company, St Louis, MO, USA) to isolate major normal fibroblasts (NFs) and CAFs [16].

Human normal ovarian epithelial cell line HOSEpiC and human OC cell lines SKOV3, SKOV3/cisplatin (cisplatin-resistant SKOV3 cells) were purchased from Cell Bank Type Culture Collection of Chinese Academy of Sciences (Shanghai, China), and A2780 and A2780/cisplatin (cisplatin-resistant A2780 cells) were provided by Shanghai Biological Technology Co., Ltd. enzyme research (Shanghai, China). Cells were cultured in Roswell Park Memorial Institute-1640 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Life Technologies, Carlsbad, CA, USA) and 1% penicillin/streptomycin (Beyotime Biotechnology Co., Shanghai, China). CAFs and NFs were co-cultured with Dulbecco’s modified Eagle’s medium (DMEM)/F12 containing 10% FBS at 37 °C in a 5% CO₂ incubator (thromo3111, Beisheng Medical Devices Co., Ltd., Jinan, China). The cells were passaged every 3 days [17].

CAFs and NFs were collected and seeded into 6-well plates coated with polylysine. Following three rinses with preheated 0.01 mol/L PBS, the cells were fixed with 4% paraformaldehyde under room temperature conditions for a 30-min period. After three PBS rinses, the cells were sealed with blocking solution (Beyotime Biotechnology Co., Shanghai, China) at 37 °C for 60 min. Afterwards, the cells were probed with rabbit antibodies against α-smooth muscle actin (α-SMA; ab32575, 1: 200), fibroblast activation protein (FAP; ab53066, 1: 50), and fibroblast-specific protein 1 (FSP1; ab124805, 1: 500) overnight at 4 °C. The antibodies were obtained from Abcam Inc., Cambridge, UK. The following day, further incubation of the cells was conducted with the following secondary antibodies: Alexa Fluor 594 conjugated donkey anti-rabbit (1: 400, A21202) and Alexa Fluor 488 conjugated donkey anti-mouse (1: 400, A21207), for 1 h in dark, which were both purchased from Life Technologies (Carlsbad, CA, USA). Subsequently, the cells were stained with 4′,6-diamidino-2-phenylindole (DAPI; Beyotime Biotechnology Co., Shanghai, China) at room temperature for 5 min, and sealed with anti-fade mounting medium (Beyotime Biotechnology Co., Shanghai, China). Finally, a high-content screening imaging system (Image Xpress Micro 4, Molecular Devices, Sunnyvale, CA, USA) was employed for photography.

Cells were lysed for 30 min using radio immunoprecipitation assay lysis buffer containing phenylmethanesulfonyl fluoride (R0010, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) on ice, and subjected to a 10-min centrifugation at 12,000 r/min at 4 °C. The total protein concentration was determined using a bicinchoninic acid kit (Pierce, Rockford, IL, USA). Next, 50 μg protein was dissolved in 2× sodium dodecyl sulfate (SDS) buffer and boiled for 5 min. Subsequently, the protein samples underwent 10% SDS–polyacrylamide gel electrophoresis and a transfer onto polyvinylidene fluoride membranes (Merck Millipore, Billerica, MA, USA) by the wet transfer method. The membrane was blocked with 5% skim milk under room temperature conditions for 1 h. An overnight incubation of the membrane was then performed at 4 °C with diluted rabbit antibodies against CD63 (ab118307, 1: 50), CD81 (ab109201, 1: 1000), tumor susceptibility gene 101 (TSG101; ab125011, 1: 1000), CDKN1A (p21) (ab109520, 1: 1000) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; ab8245, 1: 10,000). Afterwards, the membrane was probed with the horseradish peroxidase-labeled secondary antibody, goat anti-rabbit antibody to immunoglobulin G (IgG; ab205719, 1: 2000) for 1 h. After a TBST rinse, the membrane was developed using enhanced chemiluminescence (BB-3501, (Amersham, Buckinghamshire, UK). Gel imaging system was used for photography, followed by analysis using the Image J software. All the antibodies mentioned were from Abcam Inc. (Cambridge, UK).

OC cells were collected upon reaching logarithmic growth state, and resuspended in order to reach a concentration of 1 × 10⁵ cells/mL. Next, 100 μL cell suspension was subjected to a 24-h incubation at 37 °C with 5% CO₂ in a 96-well plate. After 24, 48 and 72 h, 10 μL CCK-8 reagent was applied to stain the cells. The optical density (OD) was measured at 450 nm with an enzyme-linked immunometric meter after 24 h.

A total of 100 μL cell suspension (1 × 10⁵ cells/mL) was seeded to 96-well plates, followed by treatment with cisplatin at variant concentrations (0, 1, 2, 4, 8 μg/mL; Selleck Chemicals, Houston, TX, USA) for 72 h. The sensitivity of OC cells to cisplatin was then evaluated via the CCK-8 assay, and IC50 was obtained.

Exosomes were labeled with PKH67 fluorescent staining solution (Sigma-Aldrich Chemical Company, St Louis, MO, USA). Briefly, 200 μg exosomes and 4 μg PKH67 staining solution were dissolved with 1 mL Dilument C solution (Beyotime, Shanghai, China), respectively, followed by slight mixing for 5 min. After the exosomes underwent a 2-h centrifugation at 100,000 g at 4 °C, the labeled exosomes were collected after another centrifugation at 100,000g at 4 °C for 2 h. After A2780 cells were plated to a 24-well plate at a density of 5 × 10⁴ cells each well, they were cultured overnight. The next day, the cells were respectively treated with PBS, CAF-derived exosomes (CAF-exo) or NF-derived exosomes (NF-exo) for 24 h. A Nikon Eclipse Ti confocal laser scanning microscope was used to observe the uptake of exosomes by A2780 cells. Next, the cells subjected to different co-culture were treated with cisplatin for 48 h.

A2780 cells were plated into 6-well plates. Upon reaching cell confluency of about 70%, the cells were treated with overexpression-negative control (oe-NC) and oe-CDKN1A plasmids with the use of Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). All the plasmids were from Ribobio (Guangzhou, Guangdong, China).

Mimic-NC and miR-98-5p mimic plasmids were transfected into CAFs using the same method described above. After 24 h of transfection, exosomes were isolated from CAFs. Next, the exosomes, including exosomes from CAFs transfected with miR-98-5p or mimic-NC (miR-98-5p-exo or NC-exo) were added into A2780 cells without transfection or A2780 cells after transfection. All the exosomes were preserved for subsequent experimentation.

Total RNA was collected using a RNeasy MiniKit (Qiagen company, Hilden, Germany). For mRNA and lncRNA, RNA was reversely-transcribed by a reverse transcription kit (RR047A, Takara Bio Inc., Otsu, Shiga, Japan). For miRNA, the obtained RNA was then reversely-transcribed into complementary DNA (cDNA) according to the protocols of the microRNA FirstStrand cDNA Synthesis (Tailing Reaction) kit (B532451-0020, Shanghai Sangon Biotechnology Co. Ltd., Shanghai, China). qPCR was then performed on a real-time fluorescence qPCR detection device (ABI7500, ABI, Foster City, CA, USA) with reference to the SYBR PremixExTaqTMII (Perfect Real Time) kit (DRR081, Takara Bio Inc., Otsu, Shiga, Japan); three duplicated wells were set for each sample. The general negative primers of miRs and the upstream primers of U6 internal reference were extracted from the microRNA FirstStrand DNA synthesis kit. The other primers synthesized by Shanghai Sangon Biotechnology Co., Ltd. (Shanghai, China) are listed in Table 1. Target gene expression was normalized to that of GAPDH, and miR-98-5p expression was normalized to that of U6. Relative expression was calculated using the 2-ΔΔCt method.

In total, 2 mL 0.6% bottom agarose (Gibco Company, Grand Island, NY, USA) was supplemented to each well of a 6-well plate. After the agarose at the bottom was coagulated, the cells were evenly suspended in 0.3% agarose at 37 °C. Next, 2 mL cell suspension was immediately added to the 6-well plate (2000 cells/well) and allowed to stand for 10 min at 4 °C. The cells were maintained in an incubator for 14 days at 37 °C. The number of formed clones (≥ 50 cells was regarded as one clone) was counted under a microscope. Three parallel wells were set up to obtain the average value.

Extracted cells were resuspended to adjust the density into 1 × 10⁶ cells/mL. Subsequently, 1 mL cells were fixed overnight at 4 °C using precooled 70% ethanol solution. Following re-suspension, 100 μL cell suspension (no less than 1 × 10⁶ cells/mL) was treated with 1 mL 50 mg/L propidium iodide (PI) staining solution contained RNAase (Sigma-Aldrich Chemical Company, St Louis, MO, USA) for 30 min in darkness. Afterwards, a 300-mesh nylon mesh was used to filter the cell suspension. Finally, cell cycle distribution was detected using a Gallios flow cytometer (Beckman Coulter, Inc., Chaska, MN, USA) at the excitation wavelength of 488 nm.

Annexin V-fluorescein isothiocyanate (FITC)/PI staining was conducted to detect cell apoptosis. The procedures used in cell apoptosis detection were the same with those used in cell cycle detection. The cells were dyed using 10 μL Annexin V-FITC as well as 5 μL PI under room temperature conditions for 15 min in darkness. Finally, cell apoptosis was tested with the use of a flow cytometer.

CDKN1A 3′ untranslated region (UTR) gene fragments were artificially synthesized, and then introduced into pMIR-reporter (Beijing Huayueyang Biotechnology, Beijing, China). The mutant form in the potential miR-98-5p binding sites was also constructed. The wild type (WT) and mutant (MUT) luciferase reporter plasmids were co-transfected into HEK293T cells along with miR-98-5p, respectively. The activities of both firefly luciferase (M1) and Ranilla luciferase (M2) were determined using a dual luciferase reporter assay kit (Promega, Madison, WI, USA). The target gene as well as the luciferase activity of gene promoter was expressed by M2/M1, and the average of three wells served as the final result.

Thirty specific-pathogen-free female BALB/c nude mice (aged 4–6 weeks) were from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China) and reared in a routine manner. A2780 cells were subjected to different culture patterns as follows: (1) 1.5 × 10⁶ A2780 cells were exposed to serum-free medium; (2) 1.5 × 10⁶ A2780 cells were cultured in serum-free medium containing 20 μg agomir-NC-exo; (3) 1.5 × 10⁶ A2780 cells were cultured in serum-free medium containing 20 μg agomir-miR-98-5p-exo. After 12 h, A2780 cells were thoroughly rinsed to remove the remaining exosomes.

The A2780 cells (2 × 10⁶ cells in 25 mL PBS) cultured in different ways were delivered into the groin of the nude mice in a separate manner, in order to develop a subcutaneous transplantation nude mouse model of OC. Subsequently, cisplatin, at a dose of 2.5 mg/kg/2 days, was administrated into the abdominal cavity of the nude mice [20] for a total of 21 times. The length as well as the width of the transplanted tumors in the nude mice was measured every 7 days utilizing a digital caliper. The volume of tumors was calculated by this formula: volume = length × width² × 0.5 [21] to plot the growth curve. After 42 days, the mice were euthanized by excessive anesthesia, and the tumors were removed, and weighed.

Data analysis was conducted utilizing the SPSS 21.0 software (IBM Corp., Armonk, NY, USA). All quantitative data were shown as mean ± standard deviation. Unpaired t test was applied to compare the unpaired data between two groups that conformed to normal distribution and homogeneity of variance. One-way analysis of variance (ANOVA) was used to compare data among multiple groups, followed by a Tukey multiple comparisons posttest. Comparisons among multiple groups at various time points were analyzed using repeated measures ANOVA, and Bonferroni post hoc test was further employed. The correlation between two indices was analyzed by means of Pearson’s correlation analysis. p < 0.05 was indicative of a statistically significant difference.

The cisplatin-treated OC dataset, GSE58470, was downloaded from the Gene Expression Omnibus (GEO) database (https://​www.​ncbi.​nlm.​nih.​gov/​geo/​), which included both cisplatin-sensitive OC data and cisplatin-resistant OC data. With |logFC| > 2 and a p value < 0.05 as screening criteria, 144 differentially expressed genes were obtained (Fig. 1a). Furthermore, the correlation between the 144 differential genes and cisplatin was analyzed on the STITCH database (http://​stitch.​embl.​de/​), and the drug-gene interaction network was constructed (Fig. 1b). The network of cisplatin-related genes were separately constructed (Fig. 1c). It was detected that there existed a direct relationship of TP53 and CDKN1A with cisplatin. Furthermore, the expression pattern of the top 4 genes in regard to core degree (PCNA, TP53, CDKN1A, CCND1) in cisplatin-resistant OC cells was determined, which demonstrated that CDKN1A expression exhibited the most significant change. In addition, RT-qPCR and Western blot analysis displayed that the expression patterns of CDKN1A in OC cell lines were as follows: HOSEpiCs > SKOV3 > A2780 > SKOV3/cisplatin > A2780/cisplatin (Fig. 1D-F), indicating that higher CDKN1A expression in cisplatin-sensitive OC cell lines as well as lower CDKN1A expression in cisplatin-resistant OC cell lines. Moreover, the IC50 of cisplatin on SKOV3, SKOV3/cisplatin, A2780 and A2780/cisplatin cell lines was detected to be 2.43 ± 0.14, 6.52 ± 0.17, 2.51 ± 0.17 and 6.65 ± 0.26 μg/mL, respectively (Fig. 1g). Therefore, in this study, the A2780 cell line at a cisplatin IC50 of 2.5 μg/mL was designated for the following experimentation.

The expression of CDKN1A was then silenced in A2780 cells. RT-qPCR and Western blot analysis showed that CDKN1A was significantly down-regulated after transfection of sh-CDKN1A (p < 0.05) (Fig. 1h–j). CCK-8 assay and colony formation assay illustrated that in both cisplatin-treated cells and cells without cisplatin treatment, cell proliferation and colony formation ability were notably enhanced after silencing CDKN1A treatment (p < 0.05). However, after cisplatin treatment, cell proliferation and the number of clones were progressively diminished in A2780 cells, which could be partially rescued after silencing CDKN1A (p < 0.05, Fig. 1k–m). As demonstrated by flow cytometry, in both cisplatin-treated cells and cells without cisplatin treatment, cell apoptotic rate in addition to the proportion of cells in the G1 phase displayed an obvious decline while the proportion of cells in the S phase had a marked elevation in the presence of silencing CDKN1A (p < 0.05). However, following cisplatin treatment, cell apoptotic rate and the proportion of cells in the G1 phase were obviously increased, while the proportion of cells in the S phase was markedly decreased, which could be partially blocked after silence of CDKN1A (all p < 0.05, Fig. 1n–q). These results suggested that silencing CDKN1A could significantly increase proliferation and decrease apoptosis of cisplatin-sensitive OC cells.

Among the miRs in CAF-derived exosomes, miR-98-5p was observed to show a high expression in the CAF-derived exosomes (Fig. 2a). Thus, we decided to explore the relationship between miR-98-5p and CDKN1A.

It was predicted that there was a binding site between miR-98-5p and CDKN1A based on TargetScan online website (Fig. 2b). As revealed by dual luciferase reporter assay, a significant decrease was displayed regarding the luciferase activity of CDKN1A-WT in response to miR-98-5p mimic (p < 0.05), whereas the luciferase activity of CDKN1A-MUT remained significantly unchanged by miR-98-5p mimic (p > 0.05; Fig. 2c). As illustrated by RT-qPCR and Western blot analysis in Fig. 2d–f, the CDKN1A expression was remarkably reduced by miR-98-5p mimic. Pearson’s correlation analysis showed that in A2780 cells, the miR-98-5p expression shared negative correlation with the expression of CDKN1A (r = -5.517, p < 0.05; Fig. 2g). Therefore, it was concluded that CDKN1A was the target gene of miR-98-5p.

Based on the observation under the microscope, CAFs and NFs were slender and fibrous. Immunofluorescence exhibited that the expression of specific marker proteins (α-SMA, FAP and FSP1) was enhanced in CAFs (Fig. 3a). Next, the exosomes were isolated from CAFs and NFs. Under the transmission electron microscopy, it was found that the isolated vesicles were round or elliptical membrane vesicles in disk-like structure, with complete capsule (Fig. 3b). Nanotrace analysis showed that the isolated vesicles were structures with an average particle size of 50-100 nm (Fig. 3c). Western blot analysis results exhibited that the isolated exosomes were positive for exosomal markers (CD63, CD81 and TSG101) (Fig. 3d). The above results demonstrated that the exosomes had been successfully isolated.

The fluorescence labeled exosomes from CAFs and NFs were co-cultured with A2780 cells. Through the observation under the laser confocal microscope, it was shown that obvious green fluorescence was observed in the cytoplasm of A2780 cells co-cultured with CAF-exo and NF-exo, with no significant difference in fluorescence intensity between co-cultured with CAF-exo and NF-exo (p > 0.05; Fig. 3e). Besides, based on RT-qPCR, the miR-98-5p expression in A2780 cells co-cultured with CAF-exo was profoundly higher than in A2780 cells co-cultured with NF-exo (p < 0.05; Fig. 3f).

A2780 cells were co-cultured with exosomes and treated with cisplatin. The results obtained from CCK-8 and colony formation assays displayed that cisplatin treatment significantly reduced the cell proliferation and the number of clones (p < 0.05). Relative to PBS treatment, NF-exo did not bring up significant changes in terms of the cell proliferation and the number of clones (all p > 0.05), while CAF-exo led to marked increases in the cell proliferation and the number of clones (p < 0.05; Fig. 3g–i).

Furthermore, based on the results of flow cytometry, after cisplatin treatment, a notable elevation was found regarding the percentage of cells in the G0/G1 phase, while the percentage of cells in the S phase was markedly reduced, and the apoptotic rate showed a notably increase (p < 0.05). Moreover, NF-exo resulted in no significant changes in terms of all the above indicators when compared with PBS treatment (p > 0.05). Furthermore, CAF-exo contributed to a marked decrease in the percentage of cells distributed in the G0/G1 phase, a notable elevation in the percentage of cells arrested in the S phase, as along with a decreased apoptotic rate (p < 0.05; Fig. 3j–m). These results suggested that CAF-derived exosomes could significantly promote cisplatin resistance in OC cells.

Exosomes were isolated from CAFs after transfection of NC-mimic (NC-exo) and miR-98-5p-mimic (miR-98-5p-exo). As illustrated in RT-qPCR, no significant difference existed in terms of miR-98-5p expression between NC-exo and exosomes from CAFs without any transfection (exo) (p > 0.05). However, miR-98-5p expression was increased significantly in miR-98-5p-exo (Fig. 4a). Subsequently, the exosomes were co-cultured with A2780 cells and then treated with cisplatin. Based on the results from CCK-8 and colony formation assays, treatment with cisplatin contributed to significant declines in the cell proliferation and the number of clones (p < 0.05). No significant changes were detected with regard to the cell proliferation and the number of clones between A2780 cells co-cultured with NC-exo and exo (p > 0.05). However, the A2780 cell proliferation and the number of clones displayed an obviously promotion following miR-98-5p-exo treatment (p < 0.05) (Fig. 4b–d).

As displayed in flow cytometry, following treatment with cisplatin, a notable increase was detected regarding the proportion of cells in the G0/G1 phase, while the proportion of cells in the S phase showed a decline, along with an elevated apoptotic rate in A2780 cells (p < 0.05). Moreover, co-culture with NC-exo resulted in no significant changes in terms of all the above indicators when compared with co-culture with exo (p > 0.05). Furthermore, miR-98-5p-exo resulted in diminished proportion of cells distributed in the G0/G1 phase, increased proportion of cells arrested in the S phase, as addition to decreased apoptotic rate (p < 0.05) (Fig. 4e–h). Collectively, it was suggested that CAF-derived exosomes overexpressing miR-98-5p could promote cisplatin resistance in OC.

Subsequently, we further investigated the mechanisms regarding the role that CAF-derived exosomes overexpressing miR-98-5p played in cisplatin resistance in OC cells. The CAF-exo was co-cultured with A2780 cells overexpressing CDKN1A, which were further treated with cisplatin. CCK-8 and colony formation assays documented that cell proliferation and the number of clones were significantly reduced in OC cells after treatment with cisplatin. Overexpression of CDKN1A led to profoundly reduced cell proliferation and the number of clones, which was antagonized by co-culture with miR-98-5p-exo (Fig. 5a–c). As detected in flow cytometry, the proportion of cells in the G0/G1 phase had a marked increase, while the proportion of cells in the S phase was decreased significantly, along with an elevated apoptotic rate in OC cells in the presence of cisplatin. Moreover, following CDKN1A overexpression, the proportion of cells in the G0/G1 phase reduced, while that of cells in the S phase increased, plus a decreased apoptotic rate in OC cells, which was abrogated by the combination of miR-98-5p-exo and oe-CDKN1A (Fig. 5d–g). As expected, CAF-derived exosomes could transfer miR-98-5p to facilitate OC cells resistance to cisplatin through targeting CDKN1A.

In order to investigate whether the CAF-derived exosomes could deliver miR-98-5p to influence the cisplatin resistance in OC via CDKN1A in vivo, we injected cisplatin into the abdominal cavity of nude mice transplanted with subcutaneous tumors. It was displayed that the volume as well as weight of tumors in the mice injected with agomir-NC-exo was notably increased relative to control mice (all p < 0.05). Moreover, the mice injected with agomir-miR-98-5p-exo had increased tumor volume and weight (Fig. 6a–c). In addition, RT-qPCR and Western blot assays were applied to assess miR-98-5p and CDKN1A expression in tumors of nude mice. miR-98-5p expression was markedly increased, while CDKN1A expression was notably decreased in the nude mice injected with agomir-NC-exo versus control mice (all p < 0.05). Agomir-miR-98-5p-exo resulted in further increase in miR-98-5p expression and decrease in CDKN1A expression (Fig. 6d–f). In conclusion, exosomal miR-98-5p could promote cisplatin resistance and downregulate CDKN1A in the xenograft model of OC.