MiR-624-5p enhances cell resistance against cisplatin via PDGFRA/Stat3/PI3K axis in ovarian cancer

Purpose: The purpose of this study was to evaluate the role of miR-624-5p in ovarian cancer. Methods: MiR-624-5p expression in ovarian cancer {OC) cell lines and normal cells (NCs) was evaluated and compared the differential miR-624-5p in OC A2780 cells and cisplatin-resistant OC cell line (A2780/DDP). CCK-8 was used to evaluate changes in cell viability of the A2780 and A2780/DDP cell lines as well as silenced miR-624-5p. Western Blot examined the Stat3 and phosphorylated Pi3k. The binding between PDGFRA and miR-624-5p was predicted on Targetscan and verified through Luciferase Reporter Assay. The role of PDGFRA in A2780/DDP by overexpressing PDGFRA was evaluated by RT-qPCR and CCK-8 assays. RT-qPCR assay also measured miR-624-5p expression responsive to different dosages of cisplatin and CCK8 examined viability levels correspondingly. In addition, the interplay of PDGFRA and miR-624-5p by combined downregulation of both miR-624-5p and PDGFRA were evaluated. Results: OC cells had higher miR-624-5p expression than NCs but lower compared to cisplatinresistant A2780/DDP cells. A2780/DDP cells had higher viability than OC cell line A2780. Stat3 and phosphorylated PI3K were activated in A2780/DDP cells. Silencing miR-624-5p led to lower viability in A2780/DDP cells. miR-624-5p expression dropped as the cisplatin concentration increased, resulting in decreasing viability respectively. Luciferase Reporter assay validated the binding of miR-624-5p and PDGFRA in A2780/DDP cells. Overexpressed PDGFRA induced lower cell viability in A2780/DDP cells. Downregulation of PDGFRA partially restored the lowered viability and inhibited Stat3 as well as phosphorylated Pi3k induced by miR-624-5p inhibitor. Conclusion: MiR-624-5p could add to the cellular resistance to cisplatin in OC in-vitro model, which indicated that it might help unveil the mystery of drug-resistance in clinical stage of ovarian cancer.


INTRODUCTION
Ovarian cancer severely affects women's health all over the world. Given its insidious onset and rapid development, mortality of ovarian cancer ranks the first in gynecologic oncology [1]. However, very few ovarian cancer patients can be detected in early stage, which is the main reason for the high death rate of the disease. surgery with chemotherapy, in which platinum and paclitaxel are the common chemotherapeutics agents [2,3]. In chemotherapy, however, most patients develop recurrence problem with tolerance to platinum, resulting in 5-year survival rate lower than 30% [4]. Unfortunately, the mechanism and factors related to resistance of ovarian cancer o treatment with cisplatin and how new targets agents can be developed for the disease have not been fully investigated.
In recent years, microRNAs have become key targets and hot spots in reversing chemical resistance of cancer cells. These are small molecules as well as non-coding RNAs with 20-24 nucleotides in length, which were first discovered in Caenorhabditis elegans [5]. Until now, more than 2500 mature microRNAs have been detected in human genome but unfortunately, the functions of most microRNAs have not been identified [6]. MicroRNAs can bind 3'UTR of targeted mRNAs to degrade or block translation process of mRNA so that microRNAs can suppress expression levels of target genes in cells [7]. Therefore, it is essential to determine the relationships between microRNAs and tumors, especially connections between cancers and resistance to drugs. Although studies about microRNAs regulating drug resistance in ovarian cancer are common, it is rare to find articles about miR-624-5p related to drug resistance in ovarian cancer.
Therefore, the purpose of this study was to evaluate the role of miR-624-5p and the functions of microRNA in the resistance of ovarian cancer to drugs.

Transfection
The A2780/DDP cells were planted in 96-well plate and cultured until 60% confluence was obtained. MiR-624-5p inhibitor or NC inhibitor (100pmol) was transfected into A2780/DDP cells with Lipofectamine™ 3000 Transfection Reagent (Invitrogen, USA) following manufacturer's instructions so as to form two different groups, NC inhibitor and miR-624-5p inhibitor groups of A2780/DDP cells. pcDNA3.1 NC or oe-PDGFRA was transfected into A2780/DDP cells forming two sub-groups, oe-NC and oe-PDGFRA. Finally, siRNA NC and NC inhibitor， siRNA NC and miR-624-5p inhibitor，si-PDGFRA and miR-624-5p inhibitor were co-transfected in the A2780/DDP cells to form a third group.

CCK-8 assay
The cells were seeded into 96-well plate with 5×10 4 cells per well. Next day, cisplatin (0, 3 and 5 µmol/l) were added into the well plate and cells were incubated at 37 o C, 5% CO2. Then, 10μl CCK-8 was added into each well at 24 hr, 48 hr and 72 hr and the optical density values were determined using microplate reader (Thermo Fisher, USA) at 490 nm 3 hr after adding CCK-8.

Luciferase report assay
Cells were planted into 96-well plate and incubated until 70% confluence was attained. Then, pCMV-Cypridina Luc Vector for Luciferase Assays (Thermo Fisher, USA) and NC inhibitor, miR-624-5p inhibitor and PDGFRA wt/mut were co-transfected into cells using Lipofectamine 3000. After transfection for 6 hr, medium was replaced and after 48 hr incubation, cells were rinsed with 1×PLB (Promega, USA) to lyse the cells. Pierce™ Gaussia-Firefly Luciferase Dual Assay Kit (Thermo Fisher, USA) was applied to detect luciferase activities

Western blot
Cells were first washed with phosphate buffered saline (PBS) three times, and then lysed with RIPA lysis buffer (Thermo Scientific™, USA) and 10μl PMSF (100 mmol/l) (Beyotime, Shanghai, China) on ice for 30 min and the mixture was centrifuged at 12000 rpm, 4 o C for 5 min. The proteins (40 μg) were extracted from the supernatant and BCA kit (Beyotime, Shanghai, China) was applied for quantification. The proteins were split with SDS-PAGE gel and transferred into PVDF membranes (Beyotime, Shanghai, China). After that, the membranes were rinsed with Pierce™ 20× TBS Buffer (Thermo Fisher, USA) and incubated with primary antibodies: Anti-Stat3 (1:1000; ab76315, Abcam, Cambridge, UK), anti-PI3K (1:1000; ab70912) and GAPDH (1:2000; ab181602) overnight at 4 o C. Next, membranes were washed with Tris buffered saline (TBS) three times and incubated with secondary antibodies marked by HRP: goat anti-mouse IgG (1:800; ab205719) and goat anti-rabbit IgG (1:800; ab205718) at room temperature for 2 hr and washed with TBS three times. Finally, gray bands of proteins were analyzed by BeyoECL Plus(Beyotime, Shanghai, China)with GAPDH as the internal reference.

Data analysis
Data were shown as mean ± SD using SPSS 19.0 (IBM, USA). Each experiment was repeated three times. Students' t-test and one-way ANOVA were applied. P<0.05 was considered to be significant.

MiR-614-5p was upregulated in cisplatinresistant OC cells and promoted cell viability and activated STAT3/Pi3K signaling
Compared to IOSE80 cell line, miR-624-5p was expressed higher in tumor cell lines, especially in A2780 cell line ( Figure 1A). Between OC cell line A2780 and drug-resistance OC cell line A2780/DDP, the expression of miR-624-5p was higher in A2780/DDP ( Figure 1B). Moreover, cell viabilities in were also evaluated in A2780 and A2780/DDP cells. Compared to tumor cell line, drug resistant cell line had higher cell viabilities ( Figure 1C). Then Stat3 activation and phosphorylated Pi3k were detected in both A2780 and A2780/DDP cell lines, which supported that Stat3 activity and PI3K phosphorylation were significantly increased ( Figure 1D). .

MiR-624-5p inhibitor could reduce cellular resistance to cisplatin and downregulation of PDGFRA reversed partially the effect brought by miR-624-5p through Stat3/PI3K signaling pathway in ovarian cancer
Since miR-624-5p was highly expressed in A2780/DDP cells and expression of PDGFRA was low compared to OC cells, correlation between miR-624-5p and PDGFRA was analyzed. In A2780/DDP cell line, miR-624-5p inhibitor suppressed miR-624-5p while downregulation of PDGFRA restored miR-624-5p expression ( Figure 4A). Moreover, Stat3 and p-PI3K were inhibited by down-regulated miR-624-5p and reactivated through suppressed in A2780 cell line ( Figure 4C). As concentrations of cisplatin grew, the cell viability induced by 0,3 to 5 umol/L cisplatin, was reduced in a dosagedependent manner ( Figure 4B,D,E). Among the three groups inside each subfigure, miR-624-5p inhibitor decreased cell viability while si-PDGFRA recovered the viability in A2780/DDP cells, suggesting that the resistance to cisplatin was decreased by miR-625-5p inhibition and increased by PDGFRA downregulation ( Figure  4B,D,E).

DISCUSSION
In the present study, the role of miR-624-5p in the cellular model in ovarian cancer was investigated by adopting cisplatin-resistant cell line to mimic the phenotype of resistance to cisplatin in ovarian patients. Results showed that miR-624-5p was upregulated in the cisplatinresistant cell line A2780/DDP compared to normal ovarian cell line, A2780, and cell viability was higher in A2780/DDP cells than A2780. Besides, Stat3/Pi3k signaling after inhibition of miR-624-5p, lower viability in A2780/DDP cells was observed, which indicated that downregulated miR-624-5p could contain A2780/DDP cell proliferation. MiR-624-5p expression decreased as the concentration of cisplatin increased. Targetscan predicted the putative binding of miR-624-5p and PDGFRA and luciferase confirmed the binding site. Thereafter, it was observed that PDGFRA expression was lower in resistant cells than normal OC cells. Cell viability was lower in the overexpressed PDGFRA A2780/DDP cells. This suggested that PDGFRA could deter cell viability in A2780/DDP cells. When the A2780/DDP cells were co-transfected with miR-624-5p inhibitor and si-PDGFRA, it was observed that the cell viability as well as Stat3/Pi3k were recovered by PDGFRA downregulation in this co-transfected group in comparison with the miR-624-5p inhibitor group possibly indicating that PDGFRA could reverse the effect produced by miR-624-5p in the resistant cells. In other words, miR-624-5p inhibition could contribute to decrease of cisplatin resistance while PDGFRA downregulation could add to cisplatin resistance. The finding that miR-624-5p targeted and inhibited PDGFRA indicates that miR-624-5p inhibition helped to curb cellular resistance to cisplatin in ovarian cancer.
Ovarian cancer has high morbidity and 5-year survival rate is only about 30% [8]. For patients with this condition, curative effects of the chemotherapy based on cisplatin that is commonly used is limited because of resistance to cisplatin which often results in recurrence, transition and high mortality of the disease [9]. Therefore, finding a potential mechanisms of cisplatin resistance in ovarian cancer is vital. MicroRNAs are endogenous single-strand noncoding RNA with 21-25 nucleotides in length, which could degrade transcription of mRNAs [10]. It was widely unveiled that miR-130a was upregulated in ovarian cancer and helped to enhance cisplatin resistance in the disease [11]. Similarly, miR-21 induce paclitaxel resistance by targeting APAF1 in cellular model of ovarian cancer [12]. Also, miR-411 is regulated by SLC27A2 and modulated chemo-resistance in ovarian cancer both in vitro and in vivo [13]. MiR-624-5p has been identified as a regulator in osteosarcoma and hepatoblastoma [14,15]. Since there has been no in-depth study into the role of miR-624-5p in ovarian cancer, RT-qPCRwas used in this study to check the relative expression in ovarian cancer cell lines, normal epithelial cell line as well as cisplatin-resistant cell line resulting in the finding miR-624-5p was highly expressed in cisplatin-resistant cells. CCK8 assay validated that upregulated miR-624-5p could add to cellular resistance to cisplatin in ovarian cancer. The predicted potential target gene, PDGFRA, was validated and through Oncomine Database (https://www.oncomine. org/), it was identified that a familiar mRNA PDGFRA, was among the 10% top under expressed in ovarian cancer based on the Human Genome U95A-Av2 Array (8603), Human Genome U95B Array (5939), Human Genome U95C Array (4699), Human Genome U95D Array (3584) and Human Genome U95E Array (5037) from Lu. Besides, results from this study identified lower expression of PDGFRA in resistant cells than OC cells. Previously, PDGF/PDGFR has close connections with several signaling pathways such as PI3K/AKT, JAK-STAT and Ras/MAPK [16][17][18]. In this study, it was discovered that overexpression of PDGFRA could suppress cellular resistance to cisplatin and the inhibition of PDGFRA could restore miR-624-5p expression in resistant cells and recover cell viability inhibited by miR-624-5p inhibitor.
Expressions of Stat3 and p-PI3K were also resumed through downregulation of PDGFRA after reduced by miR-624-5p inhibitor. STAT3 has been known to play an oncogenic role in ovarian cancer. It was recently discovered that STAT3 inhibitor could alleviate the resistance to cisplatin in ovarian cancer both in cellular model and mice model, suggesting that the treatment of ovarian cancer might be improved if Stat3 inhibitor is applied together with cisplatin [19]. STAT3 is a canonical signaling which regulates cell proliferation and apoptosis as well as metastasis in various cancers including ovarian cancer [20,21]. Pi3k signaling modulates cellular proliferation and apoptosis and is involved in platinum resistance in ovarian cancer by regulating miRNAs and mRNAs [22,23]. Our results agreed with these findings and enriched the regulatory mechanisms beneath Stat3/Pi3k signaling pathway in ovarian cancer.

CONCLUSION
MiR-624-5p was upregulated and targeted PDGFRA while PDGFRA was downregulated in cisplatin resistant ovarian cancer cells. MiR-624-5p inhibitor was exposed to decrease cellular resistance against cisplatin and PDGFRA inhibition could restore the cellular resistance. Stat3/Pi3k signaling was inhibited by miR-624-5p inhibition and activated by si-PDGFRA. These findings implied that downregulation of miR-624-5p might suppress the cellular resistance to cisplatin by targeting PDGFRA via Stat3/ Pi3k signaling pathway, which suggested that there might be a possibility that miR-624-5p inhibitors could be combined with cisplatin in the treatment of ovarian cancer in clinical stage.