Background
Among all gynecologic malignancies, ovarian cancer is the primary cause of death. Though the therapy of ovarian cancer has been promoted, the current treatment was limited due to the occurrence of chemotherapy-resistant cancer cells [
1]. The lack of an obvious, particular clinical presentation in the early stage and the deficiency of screening tool caused that the patients with ovarian cancer could only be diagnosed at more advanced stages. Current treatment could not cure the disease, and the 5-year survival rate of ovarian cancer is 45% [
2]. According to the demonstration of previous studies, some of the risk factors had been verified, such as family history of breast cancer [
3], circulating vitamin D [
4] and central adiposity [
5]. In recent years, several microRNAs (miRNAs) have been verified in human diseases, and many extant studies have unraveled that miRNAs were involved in ovarian cancer progression, such as miR-200 [
6], miR-15a, miR-16 [
7] and miR-506 [
8].
MiRNAs are known as small non-coding RNAs and generally deregulated in cancers. As one of the miRNAs, miR-205 is usually silenced in advanced stage of cancers [
9]. Additionally, the impacts of miR-205 on human diseases such as advanced pancreatic cancer [
10], head and neck squamous cell carcinoma [
11] and primary cutaneous T-cell lymphomas [
12] have been assessed. Beyond that, Li et al. have found in their study that miR-205 was implicated in the development of ovarian cancer [
13]. Wei et al. have found that miR-205 expression increased in ovarian cancer tissues and ovarian cancer cell lines, and overexpressed miR-205 could accelerate the invasion of ovarian cancer cells [
14]. Furthermore, Wang et al. have reported that plasma miR-205-5p significantly overexpressed in ovarian cancer patients in comparison with normal controls [
15]. In addition, it has been demonstrated by Zhang et al. that the sequence divergent miR-205 was independently overexpressed in mesenchymal-like ovarian cancer cells [
16]. These data suggested that miR-205 is highly expressed in ovarian cancer, and the expression of miR-205 was positively related to the proliferation, invasion, and migration of ovarian cancer cells. As for the target relation between miR-205 and vascular endothelial growth factor A (VEGFA), a previous study has mentioned that miR-205 was up-regulated in ovarian cancer cells exposed to VEGF, and miR-205 promoted the invasion and proliferation of ovarian cancer cells [
17]. Moreover, exosomes are ceramide-enriched vesicles, with a diameter of 40–100 nm, developed by endosomal membrane inward budding, and were secreted from the fusion of multivesicular endosomes and the plasma membrane. Exosomes could transmit signaling factors as well as miRNAs that mediate intercellular communication [
18], and the impact of exosomes on the progression of blood-based ovarian cancer has been discussed [
19]. However, the relation among miR-205, exosomes as well as ovarian cancer has not been studied yet, hence, our research was carried out to investigate the capacity of miR-205 and exosomes in ovarian cancer, and we inferred that miR-205 may act as a proto-oncogene in the development of ovarian cancer. Exosomes from donor ovarian cancer cell SKOV3, miR-205 and VEGFA could participate in the progression of ovarian cancer.
Materials and methods
Ethics statement
The study was ratified by the Ethics Committee of Jiangxi Maternal and Child Health Hospital and based on the ethical principles for medical research involving human subjects of the Helsinki Declaration. Informed written consent was obtained from all the patients.
Study subjects
A total of 80 patients with ovarian cancer (with the age of 20–72 years old) who have undergone surgical treatment in the department of gynaecology in Jiangxi Maternal and Child Health Hospital from June 2017 to June 2018 were collected as a case group. The specimens of whom were all confirmed by histopathology. The specimens were staged on the basis of the criteria of surgical-pathological stage of Federation International of Gynecology and Obstetrics (FIGO): 28 cases at I stage, 18 cases at II stage, 32 cases at III stage and 2 cases at IV stage; pathological types: 48 cases of serous carcinoma, 20 cases of clear cell carcinoma, 10 cases of mucinous carcinoma and 2 cases of endometrioid carcinoma. A number of 80 healthy females who were at the matching age with patients in the case group were randomly collected as a control group. Patients in the case group have not been treated with radiotherapy, chemotherapy or hormonotherapy before the surgery. The morning fasting venous blood (5 mL) of all subjects were centrifuged and the serum was preserved at − 70 °C for the following experiments.
Cell culture
Human ovarian cancer cell line SKOV3 was obtained from the cell resource center of Shanghai Institute of Nutrition and Health, Chinese Academy of Sciences (Shanghai, China) and was generally cultured in an incubator at 37 °C and 5% CO2, then incubated in RPMI-1640 medium containing 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA) with a concentration of 100 U/mL of penicillin and 100 μg/mL of streptomycin. The density of adherent SKOV3 cells was observed under an inverted microscope, and the cells were passaged when the cell confluence reached 90%. Cells in the logarithmic growth phase were harvested for the follow-up experiments.
Exosome separation and identification
The supernatant was collected after a 48-h cell culture. Based on a Ref. [
20], the exosomes were extracted by gradient centrifugation of the supernatant (300 g for 10 min, 1200 g for 20 min, 10,000 g for 30 min) at 4 °C, then the supernatant was centrifuged at 100,000
g at 4 °C for 1 h, and the sediments were exosomes, which were rinsed by phosphate buffered solution (PBS), then centrifuged at 100,000
g at 4 °C for 1 h and the sediments were resuspended by PBS and filtered by a 0.22 μm filter to obtain the exosome initial solution, which was preserved at − 80 °C for the following experiments. The morphology and size of exosomes were observed under an electron microscope, and its related protein expression was assessed by Western blot analysis.
Exosome uptake experiment
The exosomes were marked by PKH67 Fluorescent Cell linker kits (Sigma, St. Louis, MO, US) according to its direction, and the exosomes marked by PKH67 were acquired. A number of (0.5–1) × 105 SKOV3 cells were seeded into 24-well plates and incubated at 37 °C, with 5% CO2. The exosomes marked by PKH67 as well as SKOV3 cells were co-cultured without light for 12 h and washed by PBS for three times, 5 min/time, then fixed by paraformaldehyde for 20–30 min, rinsed by PBS for three times, 5 min/time; the nuclei were stained by 2,4-diamino-5-phenylthiazole (DAPI) (Beyotime Biotechnology Co., Ltd., Shanghai, China) for 5 min, rinsed by PBS for three times (5 min/time), and fixed. The distribution of fluorescence was observed by a laser scanning microscope (Nikon Co., Ltd., Tokyo, Japan).
The role of GW4869 inhibitor in exosome development
Cells in the logarithmic growth phase were seeded onto 24-well plates at 1 × 105 cells/well and incubated. The 24-well plates seeded with SKOV3 cells were took out 24 h in advance with medium discarded, then added with 14.5 μL GW4869 storage solution, 1.5 μL dimethyl sulfoxide (DMSO) solution and RPMI-1640 complete culture solution containing 10% FBS, making the concentration of GW4869 in each well reached 10 μM, and cells supplemented with 0 μM GW4869 were taken as the Mock group. After 48-h incubation, the total RNA was extracted from the treated cells, and miR-205 expression in supernatant and cells was evaluated using reverse transcription quantitative polymerase chain reaction (RT-qPCR).
Cell grouping and transfection
Ovarian cancer cell line SKOV3 in the logarithmic growth phase was adopted and the cells were separated into three groups: the blank group: cells without transfection; the mimics negative control (NC) group: cells transfected with miR-205 mimics NC or Cy3-mimics NC; the miR-205 mimics group: cells transfected with miR-205 mimics or Cy3-miR-205 mimics. Cy3-miR-205 mimics, Cy3-mimics NC, miR-205 mimics and mimics NC were all obtained from Guangzhou RiboBio Co., Ltd. (Guangdong, China). Cy3-miR-205 mimics, Cy3-mimics NC or miR-205 mimics and mimics NC were transfected by Lipofectamine™ RNAiMAX (Invitrogen, Carlsbad, CA, USA) on the basis of the kit instruction.
Establishment of cell co-culture models
SKOV3 cells that have transfected with Cy3-miR-205 mimics and Cy3-mimics NC for 36 h (the current SKOV3 cells were donor cells) were collected and seeded at 1 × 105 cells/well in the apical chamber of the transwell plate (the membrane pore size was 0.4 μm), the complete medium was made up to 300 μL. The basolateral chamber was seeded with generally cultured SKOV3 cells (the current SKOV3 cells were receptor cells) 1 day in advance and at 1 × 105 cells/well, three wells were set in each group. After 24-h culture of the cells in both apical chamber and basolateral chamber, the entry of Cy3-miR-205 mimics and Cy3-mimics NC into receptor cells was observed by a FSX100 intelligent biological navigator (Olympus, Tokyo, Japan); the receptor cells were harvested and the total RNA was extracted, then miR-205 expression in the receptor cells was measured by RT-qPCR. The exo-blank group: donor SKOV3 cells without any transfection were co-cultured with receptor SKOV3 cells; the exo-mimics NC group: donor SKOV3 cells were transfected with Cy3-mimics NC and co-cultured with receptor SKOV3 cells; the exo-miR-205 mimics group: donor SKOV3 cells were transfected with Cy3-miR-205 mimics and co-cultured with receptor SKOV3 cells.
5-Ethynyl-2′-deoxyuridine (EdU) assay
The donor SKOV3 cells and receptor SKOV3 cells were co-cultured for 24 h and the DNA replication ability of receptor SKOV3 cells in each group was measured by Cell-light EdU kit (Ribo Bio Co., Ltd., Guangzhou, China). The steps are as follows: the cells of each group were incubated by 100 μM EdU solution for 2 h and fixed by 4% paraformaldehyde for 20 min, then 2% glycine was added for 15-min incubation, the cells were washed by PBS two times and added with 150 μL penetrant for permeabilization, and the following steps were conducted under the guide of the direction of EdU kit. Five fields of view were randomly photographed by a fluorescence microscope (FSX100, Olympus, Tokyo, Japan), the blue fluorescence represented for all the cells, and the red fluorescence represented for the replicating cells that have been penetrated by EdU. The proliferation rate of receptor SKOV3 cells was counted as the number of cells with blue fluorescence/the number of cells with red fluorescence × 100%.
Flow cytometry
Cell cycle detection
The receptor SKOV3 cells that have been co-cultured for 24 h were collected and resuspended by PBS, the cell concentration was adjusted to 1 × 106 cells/mL and the cells were made into single cell suspension, centrifuged at 2000 rpm for 5 min to remove the supernatant. Each group was added with 500 μL 70% cold ethanol and fixed at 4 °C for 2 h. With the stationary liquid discarded, the cells were added with 1 mL PBS and centrifuged at 2000 rpm for 3 min; the cells were added with 100 μL RNase A (100 μg/mL) for water bath at 37 °C for 30 min, stained with 400 μL propidium iodide (PI) (50 μg/mL) at 4 °C for 30 min without light exposure, then detected by the machine, and the red fluorescence at 488 nm excitation wavelength was recorded.
Apoptosis detection: after 24-h co-culture, the receptor SKOV3 cells in each well were added with 1 mL serum-free RPMI-1640 medium, then 24-h starvation was followed by trypsinization of trypsin without ethylene diamine tetraacetic acid (EDTA). Subsequently, the cells were collected after 5-min centrifugation at 2000 rpm, washed twice by pre-cold PBS and centrifuged. After resuspended in 500 μL binding buffer, cells in each well were added with 10 μL AnnexinV-FITC (20 μg/mL) and 5 μL PI (20 μg/mL) for fluorescence labeling, then mixed up and incubated for 15 min. The cell apoptosis was evaluated by flow cytometry in 1 h. Judgment of the results: AnnexinV was taken as abscissa axis and PI wad taken as vertical axis; cells in the left upper quadrant were mechanical damaged cells; cells in the right upper quadrant were advanced apoptotic or necrotic cells; cells in the left lower quadrant were negative normal cells; cells in the right lower quadrant were viable apoptotic cells.
Cell migration experiment
The receptor SKOV3 cells that have been co-cultured for 24 h were collected and resuspended by serum-free RPMI-1640 medium, then seeded into the apical chamber of transwell at a density of 5 × 103 cells/well, the medium was made up to 150 μL and the basolateral chamber was added with 600 μL RPMI-1640 complete medium without antibiotic. After 24-h culture at 37 °C and 5% CO2, the cells were fixed by 95% ethanol for 10 min, stained by crystal violet dye for 20 min, then observed by an intelligent biological navigator (Olympus, Tokyo, Japan), five random fields of view were adopted and the number of transmembrane cells were counted.
Cell invasion experiment
Matrigel was melted at 4 °C overnight and diluted by pre-cold serum-free RPMI1640 medium (at a ratio of 8: 1); the medium (50 μL) was paved on the transwell polycarbonate membrane with a pore diameter of 8 μm, making all the wells were covered by Matrigel at 37 °C for 2 h. The receptor SKOV3 cells that have been co-cultured for 24 h were collected and resuspended by serum-free RPMI-1640 medium, then seeded into the apical chamber of transwell at 1 × 105 cells/well, the medium was made up to 150 μL. The basolateral chamber was added with 600 μL RPMI-1640 complete medium containing 50% FBS. After 24-h culture at 37 °C and 5% CO2, the cells were fixed using 4% paraformaldehyde for 15 min, stained by crystal violet dye for 10 min, five random fields of view were photographed and the number of transmembrane cells was recorded.
RT-qPCR
The total RNA was extracted by SunShine Bio™ kit (SunShine Bio Co., Ltd., Nanjing, China). The total RNAs of the cells and the exosomes were extracted by Trizol (Invitrogen, Carlsbad, CA, USA). The concentration and purity of RNA (the ratio of A260/A280) were evaluated on NANODROP 2000C (Thermo Fisher Scientific Co., Ltd., MA, USA), the ratio of RNA purity at 1.8–2.0 was eligible. The primers of miR-205 and U6 as well as PCR primers were all purchased from Guangzhou RiboBio Co., Ltd. (Guangdong, China), the primer sequence was not provided for business factors. The reverse transcription of VEGFA and β-actin was conducted by MMLV kit (Invitrogen, Carlsbad, CA, USA), PCR primers were synthetized by the Shanghai branch of Invitrogen (Shanghai, China). VEGFA primers are as follows: Forward: 5′-ACGGATCCATGGCGGTCCCACGTC-3′, Reverse: 5′-TTGAATTCTTACCGCCTCGGCTTGTCAC-3′. β-actin primer are as follows: Forward: 5′-ATCCGCAAAGACCTGT-3′, Reverse: 5′-GGGTGTAACGCAACTAAG-3′. The data were analyzed using 2−ΔΔCt method.
Western blot analysis
Proteins of the cells and the exosomes were extracted and the concentration was measured according to the instruction of bicinchoninic acid kit (Boster Biological Technology Co., Ltd., Wuhan, Hubei, China), the extracted proteins were added with buffer and boiled at 95 °C for 10 min (30 μg/well). Then electrophoresis separation was conducted by 10% polyacrylamide gel (Boster Biological Technology Co., Ltd., Wuhan, Hubei, China), the proteins were transferred onto polyvinylidene fluoride (PVDF) membrane and sealed with 5% bovine serum albumin (BSA) for 1 h, added with primary antibodies CD9 (1: 2000), CD63 (1: 1000), TSG101 (1: 1000), VEGFA (1: 10,000), E-cadherin (1: 1000), Vimentin (1: 1000) and β-actin (1: 5000, all from Abcam, Cambridge, UK), PCNA (1: 1000), cyclin D1 (1: 1000, both from Santa Cruz Biotechnology, Santa Cruz, CA, USA), p-AKT (1: 1000), AKT (1: 1000), p-mTOR (1: 1000), mTOR (1: 1000), Bax (1: 1000), Bcl-2 (1: 1000, all from Cell Signaling Technology, Beverly, MA, USA), MMP-2 (1: 1000), and MMP-9 (1: 1000, both from Proteintech, Chicago, Illinois, USA), and incubated at 4 °C overnight, and rinsed by tris buffer solution with tween (TBST), 3 times/5 min. The relative secondary antibodies (Southern Biotech Co., Ltd., AL, USA) were incubated for 1 h and developed by chemiluminescence reagent. β-actin was taken as an internal reference. The proteins were developed by Gel Doc EZ imager (Bio-rad, CA, USA), and Image J software was used for the grey value analysis of the target band.
Dual-luciferase reporter gene assay
The online prediction software
http://www.targetscan.org was adopted to verify the target sites of VEGFA and miR-205, the sequence was designed and synthetized by GenScript Co., Ltd. (Nanjing, China). The obtained target products as well as pMIR-REPORT™ Luciferase carrier vector were conducted with double digestion, and digestion products of restriction enzyme Hind III and Spe I were recycled, then connected by T4 DNA ligase. The Escherichia coli DH5α and competent cells were transformed, and the plasmid was extracted, then the right recombinant plasmid was acquired. SKOV3 cells were seeded onto 12-well plates at 1 × 10
5 cells/well, which were co-transfected with recombinant plasmid and miR-205 mimics for 48 h with medium discarded, each well was added with 100 μL cell lysate for 30 min, then 20 μL cell lysate was added with 100 μL LARII and the fluorescence value (A) was measured. The cell lysate was added with 100 μL Stop & Glo reagent and the fluorescence value (B) was detected; fluorescence value (A) was taken as an internal reference, and the luciferase activity value C = B/A.
Statistical analysis
All data analyses were conducted using SPSS 21.0 software (SPSS, Inc, Chicago, IL, USA). All data was subjected to normal distribution and homogeneity of variance test. The measurement data conforming to the normal distribution were performed as mean ± standard deviation. The unpaired t-test was performed for comparisons between two groups and one-way analysis of variance (ANOVA) was used for comparisons among multiple groups, the least significant difference method (LSD) test was use for pairwise comparisons. P value < 0.05 was indicative of statistically significant difference.
Discussion
The recurrence of ovarian cancer was a main scientific and clinical barrier to cancer control. Few symptoms could perform in the early stages of ovarian cancer, and most of the ovarian cancer patients were diagnosed with ovarian cancer in advanced stages [
7]. What has been verified is that the miRNAs, that were characterized as small non-coding RNAs, played a part of leading molecules in the RNA silencing [
21]. Moreover, a study has unraveled that miR-205 performed an ectopic expression in another gynecologic malignancy, endometrial cancer [
22]. Interestingly, the up-regulation of miR-205 has also been identified in ovarian cancer according to a recent study [
13]. Nevertheless, there is little known about miR-205 and exosomes in the progression of ovarian cancer. Consequently, this study was determined to assess the impacts of miR-205 and exosomes on ovarian cancer cells, and we have found that miR-205 may act as a proto-oncogene in ovarian cancer development. Exosomes from donor SKOV3 cells shuttled miR-205 could participate in the proliferation, migration, invasion, apoptosis as well as EMT progression of receptor SKOV3 cells through targeting VEGFA.
One of the most vital findings in our study suggested that miR-205 was up-regulated in ovarian cancer. Except for the diseases mentioned earlier, the high expression of miR-205 has also been proved in other human diseases, such as inflammatory breast cancer [
23] and endometrial carcinoma [
24]. Furthermore, it was obvious in our research that overexpressed miR-205 could promote proliferation and attenuate apoptosis of ovarian cancer cells. In consistent with this result, Hong Xie et al. have revealed that overexpressed miR-205 in human cervical cancer cells has the ability of increasing the cell proliferation [
25]. Another study has provided evidence to prove that overexpressed miR-205 could promote the proliferation and decrease apoptosis of nasopharyngeal carcinoma cells [
26], which is in line with the outcomes of this research. A recent study has revealed that miR-205 participated in in EMT process [
16]. Jin et al. have unveiled that the overexpression of miR-205 inhibited E-cadherin expression and promoted Snail expression in endometrial carcinoma cells [
27]. Moreover, Wang et al. have discovered that miR-205 inhibitor could increase the expression of E-cadherin and decrease the expression of Snail2 and Vimentin [
28], and it has been unraveled that miR-205 overexpression could down-regulate E-cadherin and up-regulated Snail expression [
26]. It has been uncovered by Duan et al. that the inhibited miR-205 could restrain the expression of MMP-2 and MMP-9 [
29]. Results of our research unearthed that after miR-205 was overexpressed, the levels of MMP-2, MMP-9, and Vimentin were elevated, and the expression of E-cadherin was reduced, which were in line with the published researches.
Another significant outcome of our study is that overexpressed miR-205 could down-modulate VEGFA expression. Similarly, the target relation between miR-205 and VEGFA has also been unraveled in patients with breast cancer [
30]. What’s more, exosomes from donor ovarian cancer cell SKOV3 shuttled miR-205 could advance the migration as wells as invasion of ovarian cancer cells via targeting VEGFA. The similar results in a previous research proved that miR-205 was implicated in the progression of ovarian cancer via targeting VEGFA, and was able to inhibit the invasion of ovarian cancer cells [
14]. The correlation between exosomal overexpressed miR-205 and EMT has been uncovered in our research that overexpression of miR-205 has the capacity to modulate the progression of EMT in ovarian cancer. Chang Xu et al. have given out confirmation that miR-205 could suppress EMT of gastric cancer cells [
31]. It has been proved that some of the patient-derived ovarian cancer effusion exosomes might have clinical relevance [
32]. Additionally, a study has revealed that the let-7 family, which is known to restrict cell proliferation, was overexpressed in exosomes from SKOV3 cells. Meanwhile, the miR-200 family, which is able to repress EMT, was only expressed in exosomes derived from OVCAR3 cells [
6]. The promotive role of VEGFA in EMT process has also been unveiled in a recent study [
33], which suggested that VEGFA could elevate the tumor-initiating stem cell population in different cancers, and also induce EMT and metastasis. All of these data have further confirmed the mechanism and function of miR-205, VEGFA and exosomes in human diseases.
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