Xiaojuan Su,Binbin Wang,Bo Zhang,Shiwen Pan
MiR-588 acts as an oncogene in ovarian cancer and increases the radioresistance of ovarian cancer cells
ovarian cancer,radioresistance,miR-588,SRSF6
Abstract The therapeutic outcomes of ovarian cancer (OVCA) patients are majorly limited by the development of acquired chemo/radioresistance and the lack of targeted therapies. Accumulating studies demonstrate that microRNAs are involved in tumorigenesis and radioresistance. This study aims to illustrate the role of miR-588 in the radioresistance of OVCA cells. The levels of miR-588 and mRNAs were detected by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). OVCA cell viability, proliferative, migratory and invasive capacities were evaluated by the cell counting kit-8 (CCK-8) assay, colony formation assay, wound healing assay and transwell assay, respectively. The luciferase activities of plasmids containing wild -type and mutant serine/arginine-rich splicing factor 6 (SRSF6) 3'-untranslated region in miR-588 silenced OVCA cells were detected by a luciferase reporter assay. We found that miR-588 was overexpressed in OVCA tissues and cells. Knockdown of miR-588 exerted an inhibitory effect on the proliferation, migration and invasion and strengthened the radiosensitivity of OVCA cells, whereas overexpression of miR-588 increased the radioresistance of OVCA cells. SRSF6 was verified to be targeted by miR-588 in OVCA cells. In addition, the expression level of miR-588 was negatively correlated with that of SRSF6 in OVCA clinical samples. Rescue assays indicated that SRSF6 knockdown reversed the effect of miR-588 inhibition of OVCA cells under radiation. Overall, miR-588 acts as an oncogene in OVCA and increases the radioresistance of OVCA cells by targeting SRSF6.
MiR-588 acts as an oncogene in ovarian cancer and increases the radioresistance of ovarian cancer cells

The therapeutic outcomes of ovarian cancer (OVCA) patients are majorly limited by the development of acquired chemo/radioresistance and the lack of targeted therapies. Accumulating studies demonstrate that microRNAs are involved in tumorigenesis and radioresistance. This study aims to illustrate the role of miR-588 in the radioresistance of OVCA cells. The levels of miR-588 and mRNAs were detected by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). OVCA cell viability, proliferative, migratory and invasive capacities were evaluated by the cell counting kit-8 (CCK-8) assay, colony formation assay, wound healing assay and transwell assay, respectively. The luciferase activities of plasmids containing wild -type and mutant serine/arginine-rich splicing factor 6 (SRSF6) 3'-untranslated region in miR-588 silenced OVCA cells were detected by a luciferase reporter assay. We found that miR-588 was overexpressed in OVCA tissues and cells. Knockdown of miR-588 exerted an inhibitory effect on the proliferation, migration and invasion and strengthened the radiosensitivity of OVCA cells, whereas overexpression of miR-588 increased the radioresistance of OVCA cells. SRSF6 was verified to be targeted by miR-588 in OVCA cells. In addition, the expression level of miR-588 was negatively correlated with that of SRSF6 in OVCA clinical samples. Rescue assays indicated that SRSF6 knockdown reversed the effect of miR-588 inhibition of OVCA cells under radiation. Overall, miR-588 acts as an oncogene in OVCA and increases the radioresistance of OVCA cells by targeting SRSF6.

Ovarian cancer (OVCA) is the major cause of gynecologic-related mortality worldwide [1]. The average annual OVCA incidence rate in the United States was 11.5 per 100 000 women from 2010 through 2014 [2]. OVCA is more commonly diagnosed in women over 50 years old [3, 4]. It is generally identified as a ‘hard-to-suspect’ cancer because it often presents with vague nonspecific symptoms [5]. The most common treatment for OVCA is surgery combined with chemotherapy and radiotherapy. Nevertheless, most OVCA patients are sensitive to radiotherapy and are prone to recurrence, which leads to the death of patients [6]. Thus, it is important to look for new strategies to improve the therapy of OVCA. MicroRNAs (miRNAs) are small noncoding RNA molecules that can bind to target mRNAs in the 3′-untranslated region (3′-UTR) of targeted genes [7, 8]. Increasing evidence demonstrates that aberrant expression of miRNAs contributes to tumorigenesis and pathogenesis in a variety of human malignancies [9, 10]. Studies suggest that miRNAs participate in various cancer processes, including proliferation, migration, apoptosis and invasion. Mohammadi et al. suggested that miRNA can be a promising therapeutic biomarker for predicting radioresistance in cancers [11]. Therefore, miRNAs represent a novel therapeutic strategy for tumor treatment, including OVCA. For example, Wang et al. found in their study that miR-27a stimulates the proliferative and migratory capacities of OVCA cells by regulating forkhead box transcription factor O1 (FOXO1) expression [12]. Yang and Ma discovered that, through the progression of OVCA, miR-584 overexpression can attenuate proliferative and migratory abilities of OVCA cells [13]. An increasing number of miRNAs are shown to be new therapeutic targets for OVCA. Radiotherapy is a commonly used method in OVCA treatment, and previous studies showed that more and more miRNAs are found to regulate the radioresistance of OVCA cells. For example, Wang et al. found that miR-4478 upregulation retards OVCA cell proliferation and sensitizes OVCA cells to radiation [14]. Zhang and Zhang discovered that miR-214 targets mRNA pentaerythritol tetranitrate (PETN) to promote radioresistance in OVCA cells [15]. In addition, the miRNA miR-588 involved in our study has been found to participate in the progression of different cancers, including colorectal cancer [16], laryngeal squamous cell carcinoma [17] and osteosarcoma [18]. We obtained from the GEO database that miR-588 expression in primary OVCA tissues was significantly different from that in recurrent tumor tissues. In this study, we focused on the function of miR-588 in regulating OVCA radioresistance, which has not been studied yet.

Fifty-one paired OVCA specimens and adjacent normal tissues were collected from 51 patients at the hospital. The fresh specimens were immediately frozen in liquid nitrogen and stored at −80°C until use. All the patients had not received any radiotherapy or chemotherapy before the surgery. Moreover, all the participants signed written informed consent, and this study was approved by the hospital ethics committee. The OVCA cell lines, SKOV-3 and A2780, and normal human ovarian surface epithelial cells (HOSEpiC) were purchased from the American Type Culture Collection (Manassas, VA, USA). A Faxitron 43855F X-ray Irradiator (Faxitron Bioptics LLC, Tucson, AZ, USA) was utilized for the radiation of cells. The radiation settings were as follows: 160 kV, 6.3 mA, for 1.1 min for the radiation dose of 0, 2, 4, 6 and 8 Gy, respectively. Next, all the cells were cultured in RPMI-1640 medium (Sigma, St. Louis, MO) containing 10% fetal bovine serum (FBS; Gibco, CA, USA), streptomycin (0.1 mg/ml) and penicillin (100 U/ml) in a humid incubator containing 5% CO2 at 37°C.

The miR-588 inhibitor, miR-588 mimics, sh-SRSF6 and their negative controls (NC inhibitor, NC mimics and sh-NC) were synthesized by Shanghai GenePharma (Shanghai, China). Lipofectamine 3000 (Invitrogen, CA, USA) was utilized to perform the transfection according to the manufacturer’s protocol. After transfection for 48 h, the cells were used for further experiments.

Total RNAs were extracted from OVCA cell lines using TRIzol reagent (Invitrogen) and then cDNA was obtained by reverse transcription. Subsequently, to detect the levels of miR-588 and mRNAs in A2780 and SKOV-3 cell lines, RT-qPCR was conducted using SYBR Green Supermix and Hairpin-it TM miRNAs qPCR kit (GenePharma) under the instructions of the manufacturer. In this assay, glyceraldehyde-3phosphate dehydrogenase (GAPDH) and U6 acted as internal control for RNA levels, and the data were calculated by the 2-ΔΔCT method. The primer sequences used in this assay are as follows: miR-588: forward 5′-GGGUUGGCCACAAUGGGU-3′. reverse 5′-GTCGTATCCAGTGCGTGT-3′. U6: forward 5′-CTCGCTTCGGCAGCACA-3′. reverse 5′-AACGCTTCACGAATTTGCGT-3′. SRSF6: forward 5′-GGTCGCGACGGCTATGATTA-3′. reverse 5′-ATCCTTCAAGTCCTGCCTGCCAGC-3′. GAPDH: forward 5′-CTGGGCTCACTGAGCACC-3′. reverse 5′-AAGTGGTCGTTGAGGGCAATG-3′.

Cell proliferative capacity of OVCA cells was evaluated by colony formation assay. About 500 cells were incubated in culture dish containing 5 ml preheated medium. Next, cells were incubated in an incubator for 2 weeks at 37°C with 5% CO2 until visible clones appeared. Subsequently, the colonies were fixed with 5 ml 4% paraformaldehyde for 30 min. After the fixation, the cells were dyed with 0.1% crystal violet dye for 30 min. Finally, the number of colonies was calculated under an optical microscope.

RIPA buffer was utilized to lyse the OVCA cells. The protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel electrophoresis and then transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Next, 5% skim milk was employed to block the membranes for 1 h at room temperature, then the membranes were incubated with primary antibodies overnight at 4°C and the primary antibodies are as follows: serine/arginine-rich splicing factor 6 (SRSF6) antibody (ab140602), family with sequence similarity 136, member A (FAM136A) antibody (ab266445), jade family PHD finger 2 (JADE2) antibody (ab161607), ariadne homolog 2 (ARIH2) antibody (ab133744), ddb1- and Cul4-associated factor 11 (DCAF11) antibody (ab221071) and GAPDH antibody (ab8245). The cells were further incubated with secondary antibody for 1 h at room temperature. In this assay, we applied SuperSignal chemiluminescent substrate (Millipore) to visualize the protein bands. GAPDH acted as a loading control.

The binding between miR-588 and SRSF6 3′UTR was confirmed by luciferase reporter assay. The wild-type (WT) and mutated type (MUT) SRSF6 3′UTR were cloned into the pmirGLO luciferase vector to construct luciferase plasmids SRSF6-WT and SRSF6-MUT. Subsequently, we transfected SRSF6-WT and SRSF6-MUT into A2780 and SKOV-3 cells together with miR-588 inhibitor and NC inhibitor using Lipofectamine 3000 (Invitrogen) under the manufacturer’s instruction. After transfection for 48 h, the luciferase activity was detected by Dual Luciferase Reporter Assay System (Promega, Madison, WI).

The CCK-8 assay was employed to measure OVCA cell viability. First, cells were plated into 96-well plates (1 × 104 cells/well) and then incubated for 24, 48 and 72 h. Next, 10 μl CCK-8 reagent (Dojindo Laboratories, Kumamoto, Japan) was added to each well for a further 2 h of incubation. Finally, OVCA cell viability was assessed using a microplate reader by measuring the optical density (OD) value at 450 nm.

A wound healing assay was conducted to evaluate the migratory ability of OVCA cells. SKOV-3 and A2780 cell lines were seeded in a six-well cell culture plate until 90% confluent. A 10 μl pipette was used to generate wound healing. Next, the treated cells were washed with PBS and cultured in FBS-free medium for 48 h. A Leica DMI6000 B Fully Automated Inverted Research Microscope (Leica Microsystems, Germany) was used to observe the scratch widths. The OVCA cells were imaged at 0 and 24 h. The healed wound distance was calculated by Image-Pro Plus software.

Transwell chambers (Millipore) were utilized for cell invasion assay. At first, A2780 and SKOV-3 cells (1 × 104) in serum-free Dulbecco's modified Eagle’s medium (DMEM) were seeded into the upper chamber coated with Matrigel (BD Biosciences, Bedford, MA, USA) for 48 h at 37°C. Subsequently, the cells invaded across the Transwell membrane were fixed with 4% paraformaldehyde for 10 min and then stained with 0.1% crystal violet for 20 min. Finally, the invaded cells were counted and examined using a microscope.

Statistical analysis was conducted using a SPSS software package (SPSS Standard version 13.0, SPSS Inc). Data are expressed as the mean ± standard deviation, and the experiments in this study were performed at least three times. P < 0.05 was considered statistically significant.

A miRNA microarray dataset (GSE135469) was obtained from GEO database (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE135469). Recurrence is the major cause of treatment failure in patients with OVCA. GSE135469 dataset shows novel miRNAs contributing to OVCA recurrence [19]. We examined miRNA expression from three pairs of primary and recurrent tumor tissues from two patients in the GSE135469 dataset using GEO2R (filter criteria: LogFC>1). The results showed that two miRNAs, hsa-miR-588 (LogFC = 2.841) and hsa-miR-503 (LogFC = 1.305), were upregulated in recurrent tumor tissues compared with primary tumors (Fig. 1A). The role of has-miR-503 in OVCA has been well characterized in previous studies [20, 21]. Therefore, we selected hsa-miR-588 as the object for the study. Next, we carried out RT-qPCR to assess miR-588 expression in OVCA tissues and cells. Results showed that miR-588 was elevated by almost 2-fold in OVCA tissues compared with adjacent tissues (Fig. 1B). In addition, miR-588 was upregulated by >3-fold in A2780 and SKOV-3 cells compared with HOSEpiC cells (Fig. 1C). Importantly, the analysis of miR-588 expression and clinicopathological characteristics of OVCA cancer showed that miR-588 expression was correlated with distant metastasis (P = 0.0010), lymph node metastasis (P = 0.0001) and FIGO stage (P = 0.0066) (Table 1).

To detect the biological role of miR-588 in OVCA cells, we conducted several loss-of-function assays. First, we use miR-588 inhibitor to knock down miR-588 expression and the RT-qPCR results revealed that the miR-588 level was significantly downregulated after transfection with miR-588 inhibitor into A2780 and SKOV-3 cells (Fig. 2A). As presented by the CCK-8 assay, OVCA cell viability was inhibited after miR-588 silencing (Fig. 2B). Colony formation assay showed decreased number of colonies after miR-588 inhibition, suggesting that miR-588 silencing suppressed OVCA cell proliferation (Fig. 2C). Furthermore, the healed wound percentage of miR-588 inhibitor transfected cells was lower than that in the NC inhibitor group, which indicated that miR-588 knockdown suppressed OVCA cell migration (Fig. 2D). OVCA cell invasion was also suppressed by miR-588 knockdown, as demonstrated by the transwell assay (Fig. 2E).

To explore the effect of miR-588 on radioresistance in OVCA cells, the following experiments were performed. First, as presented by RT-qPCR, expression of miR-588 was decreased after radiation as time goes by (Fig. 3A). Colony formation assay showed that miR-588 inhibitor significantly reduced the survival fraction of OVCA cells under radiation of 6 and 8 Gy compared with NC inhibitor (Fig. 3B), suggesting that inhibition of miR-588 might increase the radiosensitivity of OVCA cells. Next, we detected whether inhibition of miR-588 strengthens the effect of irradiation on cell migration and invasion. In contrast with 8 Gy of irradiation, 6 Gy of irradiation had a moderate inhibitory effect on cell survival fraction. We thus chose 6 Gy of irradiation for the following experiments. The results of RT-qPCR showed that miR-588 expression was significantly downregulated by 6 Gy of irradiation or miR-588 inhibitor, while it hardly changed in the 6 Gy of irradiation + miR-588 inhibitor group compared with the miR-588 inhibitor group (Fig. 3C). After incubation for 24 h, the healed wound percentage of cells under 6 Gy of irradiation or miR-588 inhibitor transfection was decreased compared with that under 0 Gy of irradiation, whereas the healed wound under treatment of 6 Gy of irradiation + miR-588 inhibitor was further decreased, indicating that miR-588 inhibition enhanced the effect of radiation on OVCA cell migration (Fig. 3D and E). Consistent with the results of wound healing assay, the transwell assay showed that the invasive capacity of OVCA cells was significantly reduced when treated with 6 Gy of irradiation + miR-588 inhibitor, compared with 6 Gy of irradiation alone or miR-588 inhibitor alone (Fig. 3F).

Next, we decided to upregulate miR-588 expression by using miR-588 mimics to determine whether cell migration and invasion increase compared to when radiation is used alone. As expected, overexpression of miR-588 significantly increased the survival fraction of OVCA cells subjected to irradiation (Fig. 4A). RT-qPCR analysis revealed that 6 Gy of irradiation downregulated miR-588 expression, and transfection with miR-588 mimics restored miR-588 expression under irradiation (Fig. 4B). The wound healing assay (Fig. 4C and D) and transwell assay (Fig. 4E) showed that 6 Gy of irradiation alone significantly reduced cell migration and invasion, and miR-588 overexpression alone promoted cell migration and invasion, compared with control. Interestingly, miR-588 overexpression significantly restored migration and invasion under 6 Gy of irradiation compared with the 6 Gy or 6 Gy + NC mimics groups, suggesting that miR-588 overexpression enhanced the radioresistance of OVCA cells.

To further investigate the regulatory mechanism of miR-588 in OVCA radiotherapy, we examined StarBase database (https://starbase.sysu.edu.cn/index.php) to identify the target genes of miR-588. The website exhibits thousands of genes that have binding sites for miR-588. We searched five genes (SRSF6, FAM136A, JADE2, ARIH2 and DCAF11) [search category: CLIP Data: strict stringency (≥5); Degradome Data: high stringency (≥3); Program Number: 3 program]. The results of RT-qPCR and western blotting demonstrated that SRSF6 mRNA and protein level were significantly upregulated in OVCA cells after inhibition of miR-588 among these candidate genes (Fig. 5A and B). Therefore, we selected SRSF6 as the research object. According to GEPIA database (http://gepia.cancer-pku.cn/), SRSF6 expresses a low level in OVCA tissues compared with normal tissues (Fig. 5C). RT-qPCR confirmed downregulation of SRSF6 expression in OVCA tumor tissues compared with control (Fig. 5D). Correlation analysis showed that miR-588 expression was negatively correlated with SRSF6 expression in OVCA tissues (Fig. 5E). In addition, a low level of SRSF6 was shown in OVCA cells (Fig. 5F). Next, the binding sites between SRSF6 and miR-588 were predicted by StarBase (Fig. 5G). The luciferase reporter assay indicated that miR-588 downregulation increased the luciferase activity of SRSF6-WT (Fig. 5H), confirming that miR-588 targeted SRSF6. In addition, RT-qPCR presented that the SRSF6 level was time-dependently increased under radiation (Fig. 5I).

To probe the influence of miR-588 and SRSF6 on radioresistance, the following assays were performed. In this part, all the experiments are performed in A2780 and SKOV-3 cells under 6 Gy of irradiation. First, the colony formation assay presented that cell proliferation suppressed by miR-588 inhibition was restored by SRSF6 knockdown (Fig. 6A). Similarly, the wound healing assay demonstrated that SRSF6 downregulation restored cell migration inhibited by miR-588 silencing (Fig. 6B). As presented by the transwell assay, under 6 Gy of irradiation, the inhibitory effect on cell invasion caused by miR-588 downregulation was reversed by knockdown of SRSF6 (Fig. 6C).

More than 50% of miRNA genes are located in cancer-associated genomic regions or in fragile sites, suggesting that miRNAs may play a more important role in the pathogenesis of a limited range of human cancers than previously thought [22]. In recent years, accumulating evidence has shown that miRNAs play a crucial role in OVCA progression [23]. Several miRNAs, such as miR-27a and miR-29c-3p, are expressed aberrantly in OVCA and perform different functions [12, 24]. Here, we focused on miR-588, which was found to be overexpressed in OVCA tissues compared with normal adjacent tissues. Studies have demonstrated that the dysregulation of miR-588 is relevant to tumor progression. Yu et al. discovered that miR-588 downregulation stimulates the proliferative, migratory and invasive capabilities of osteosarcoma cells [18]. A previous study demonstrated that miR-588 overexpression represses gastric cancer cell invasion and migration [25]. In our study, we conducted wound healing assay and transwell assay and found that miR-588 silencing reduced migratory and invasive abilities of cells. Radiation could suppress the expression of miR-588, and miR-588 downregulation could enhance the radiosensitivity of OVCA cells, while miR-588 overexpression increased the radioresistance of OVCA cells. Data from our study suggest that miR-588 might be a novel target for radiosensitization in OVCA. It has been well known that miRNA inhibits target gene expression in a posttranslational mechanism [26]. In this exploration, we found that SRSF6 was the target gene for miR-588, and data from a luciferase reporter assay demonstrated that miR-588 is directly bound to SRSF6 3′UTR. SRSF6 modulates the cellular processes of different cancer cell lines. For example, Si et al. studied that SRSF6 overexpression can reverse the inhibited cell proliferation and the induced cell apoptosis caused by ZNF561-AS1 knockdown [27]. In pancreatic cancer, SRSF6 is targeted by miR-193a-5p, and SRSF6 downregulation contributes to the metastasis of pancreatic cancer cells [28]. Although SRSF6 has been known to regulate the biological processes of various cancers, including colorectal cancer, breast cancer and small-cell lung cancer [29–31], the involvement of SRSF6 in OVCA has not been reported. This study showed that SRSF6 expression was induced by radiation in OVCA cells. In addition, the knockdown of SRSF6 rescued the inhibited OVCA cell proliferation, metastasis and invasion caused by miR-588 inhibition. In conclusion, this study demonstrated that miR-588 silencing reduced the radioresistance of OVCA cells and miR-588 overexpression strengthened the radioresistance of OVCA cells. Moreover, miR-588 increases the radioresistance of OVCA cells by targeting SRSF6. Our results suggested that the miR-588/SRSF6 axis might be a potential target to improve the response of OVCA to radiation therapy.

X.S. and B.W. designed this study. B.Z. collected and analyzed the data. S.P. interpreted the data and revised the manuscript. All authors have read and approved the final manuscript.

The authors declare that there are no competing interests in this study.

This work was supported by Social Development Guiding Program of Suzhou City in China (No. SYSD2019110) and the Project of State Key Laboratory of Radiation Medicine and Protection, Soochow University (No. GZK1202009).

Data sharing is not applicable as no datasets were generated or analyzed in this review article.