circBIRC6 regulates cisplatin resistance of choroidal melanoma via positively modulating ERK by sponging miR-503-3p

Background/Aims circRNA plays a key regulatory role in various human tumors. This study was carried out to investigate the molecular mechanism of circBIRC6 in the regulation of choroidal melanoma cisplatin (DDP) resistance. The expression of circBIRC6 in choroidal melanoma tissues and cells (MUM-2B, MUM-2B / DDP) was detected by RT-qPCR. The function of circBIRC6 in DDP resistance of choroidal melanoma was analyzed by functional loss and overexpression experiments. DDP inhibition rate was detected by MTT method. Western blot was used to analysis the protein levels of P-gp, MRP1 and ERK. The relationship between circBIRC6, miR-503-3p and ERK was analyzed by CircRIP and luciferase reporter vector assay.


Abstract
Background/Aims circRNA plays a key regulatory role in various human tumors. This study was carried out to investigate the molecular mechanism of circBIRC6 in the regulation of choroidal melanoma cisplatin (DDP) resistance.

Methods
The expression of circBIRC6 in choroidal melanoma tissues and cells (MUM-2B, MUM-2B / DDP) was detected by RT-qPCR. The function of circBIRC6 in DDP resistance of choroidal melanoma was analyzed by functional loss and overexpression experiments. DDP inhibition rate was detected by MTT method.
Western blot was used to analysis the protein levels of P-gp, MRP1 and ERK. The relationship between circBIRC6, miR-503-3p and ERK was analyzed by CircRIP and luciferase reporter vector assay.
Result circBIRC6 was up-regulated in choroidal melanoma, and it was signi cantly raised in choroidal melanoma tissues and cells that are resistant to DDP. CircBIRC6 regulated DDP resistance of choroidal melanoma cells by binding to miR-503-3p. ERK was directly targeted by miR-503-3p, and ERK inhibited miR-503-3p-induced DDP resistance. Finally, the expression of circBIRC6 was positively correlated with ERK, and it was veri ed that circBIRC6 regulated ERK by targeting miR-503-3p.

Conclusion
CircBIRC6 regulated the expression of ERK choroidal melanoma cells and DDP resistance via miR-503-3p.
These results suggested that the knockout of circBIRC6 may provide an effective treatment strategy for choroidal melanoma.

Background
Choroidal melanoma is the most common type of uveal malignant tumor, and it is also a common intraocular malignant tumor in adults. It is mainly derived from melanocytes, with high malignancy and mortality [1]. Its incidence is second only to that of retinoblastoma in children, and it is considered as the "number one killer" in ophthalmic diseases [2]. Studies have shown that its incidence has increased in recent years [3]. At present, according to the demands of patients, their own vision and general conditions, the treatment mainly includes transpupillary thermotherapy and local resection. Most patients with choroidal melanoma are already at the advanced stage at the time of treatment, and platinum-based chemotherapy is the majority of patients with advanced choroidal melanoma [4]. Cisplatin (DDP) is the most common platinum-based rst-line chemotherapy drug [5]. Its main mechanism of action is to inhibit DNA replication, affect cell transcription and translation, and promote tumor cell apoptosis [6]. Patients resistant to cisplatin is an important factor affecting the survival prognosis of patients with treatment failure [7]. In-depth study of the molecular mechanism of drug-resistant tumor cells may help us to understand the proliferation and metastasis of tumor cells, and obtain the ability to avoid programmed cell death.
Circular RNA (circRNA) is involved in regulating the expression level of genes on multiple levels such as epigenetics, transcription level, post-transcription level, and the occurrence and development of tumors [8].
It has been found that circRNA is widely involved in genome regulation, including X-chromosome silencing, transcriptional interference or activation, genome imprinting, chromatin modi cation, intranuclear transport and other important life processes, and is closely related to many diseases such as malignant tumors [9,10]. With the discovery of new circRNAs and the gradual recognition of their functions, the role of circRNAs in tumor pathogenesis and drug resistance has become a hot topic in the eld of tumor research [11]. Studies have shown that circBIRC6 is expressed in a variety of malignant tumors, and can regulate the biological processes of related tumors, such as liver cancer [12]. However, its effect on cisplatin resistance in tumor cells has not been reported. Therefore, it is very important to explore the mechanism of cisplatin tolerance of choroidal melanoma cells by circBIRC6.
CircRNA regulate tumor resistance by a variety of mechanisms, which can affect mRNA expression or competitively bind miRNA to affect the expression of downstream target genes [13]. MiRNA are involved in important life processes such as cell proliferation and apoptosis, gene expression regulation, and individual development [14]. miRNA is closely related to tumorigenesis, development, and resistance to chemotherapy [15]. MiR-503-3p is a newly discovered mi RNA that is involved in the proliferation of certain tumors [16]. For example, research has found that miR-503-3p promotes epithelial-mesenchymal transition in breast cancer [17]. Studies found that the expression level of miRNAs is up-regulated or down-regulated, which directly causes the abnormal expression of target gene protein, and ultimately changes the drug sensitivity of tumor cells through cell signaling pathways [18]. MAPK signaling, including extracellular signal-regulated protein kinases (ERK) and c-Jun NH-terminal kinase (JNK) and stress-activated p38 MAP kinasep38/SAPK, is very important for regulating the key functions (apoptosis, differentiation) of tumor cells [19]. Targeting these signaling pathways can induce apoptosis. Several studies have reported the treatment of cisplatin leads to the activation of ERK in several cancers [20]. The aim of this study was to elucidate the mechanism of cisplatin resistance regulated by circirc6 through

Quantitative reverse transcription PCR (qRT-PCR)
Total RNA in tissues and cells was extracted using TRIzol reagent (Biosntech, Beijing, China). The quality of RNA was analyzed using NanoDrop 1000 (NanoDrop Technologies; Thermo Fisher Scienti c, Inc.).
SYBR-Green (Takara Biotechnology, Co., Lt. (Dalian, China) was used in by qRT-PCR. Ampli cation was performed by ABI 7,500 real-time PCR system. A qScript microRNA cDNA synthesis kit (Quantabio, Beverly, California, USA) was used for cDNA synthesis. The expression levels of circBIRC6 and miR-503-3p were calculated using the2-△△CT method. The expression levels of miRNA were standardized by U6 [21]. The primer sequences were as follows: DDP sensitivity assay SGC7901 cells and SGC7901/DDP cells of blank group, NC group, transfection group were inoculated into 96 well plates with 5000 cells/well respectively, and cultured at 37 ℃, 5% CO 2 for 24 hours in saturated humidity environment. After 24 hours of culture, the original culture solution was aspirated, and the cisplatin-containing culture solution with a nal concentration of 0, 2, 4, 8, 16, 32 μmol/L was added [22]. The cells were not inoculated, but only the holes with medium were used as the zeroing holes, and the holes without medicine were used as the control group. Each concentration was set with 5 multiple holes, after 48 h of culture, the absorbance at 490 nm was detected.

CircRIP
The biotin-labeled circBIRC6 probe was designed and synthesized by Gene Pharma (Shanghai, China). The cells were seeded in a petri dish for 4 hours. Next, the cells were transfected with a speci c biotinlabeled probe or control probe (200 nM) for 24 hours. It was incubated with formaldehyde for 10 minutes and terminated with a glycine solution.The supernatant was centrifuged. After mixed with streptavidinlabeled magnetic beads (M-280, Invitrogen, CA (USA, USA), it was incubated overnight. Then, the mixture was washed and reverse-crosslinked by lysis buffer containing proteinase K. Finally, miRNeasy Mini Kit (Qiagen, Dusseldorf, Germany) was used for subsequent quantitative detection of total RNA.

Statistical methods
The monitoring data were analyzed by SPSS19.0 statistical software. The results of data analysis were represented as mean ± standard deviation (mean ±SD). Multigroup data analysis was founded on oneway ANOVA. LSD test was used for subsequent analysis. P < 0.05 indicated the difference was signi cant.  <0.05, Fig.2D, 2E). These results indicated that circBIRC6 played an key role in the regulation of DDP sensitivity in choroidal melanoma cells.

miR-503-3p was targeted by circBIRC6
We predicted with online prediction tool Starbase v2.0 and miR-503-3p was identi ed as a potential target for circBIRC6 (Fig.3A). In addition, it was found that miR-503-3p expression level was signi cantly reduced in the DDP-sensitive group and DDP-resistant group contrasted with that in the Normal group (P <0.05), and miR-503-3p expression level was decreased more signi cantly in the DDP-resistant group ( Figure 3B). Contrasted with the CM group, the miR-503-3p expression level in the MUM-2B group and MUM-2B/DDP group were signi cantly decreased (P <0.05), and the miR-503-3p expression level in the MUM-2B/DDP group was decreased more signi cantly (Fig.3C). The expression level of circBIRC6 was negatively correlated with miR-503-3p in choroidal melanoma tissues (Fig.3D). And the luciferase activity was signi cantly reduced in miR-503-3p mimic and circBIRC6-WT co-transfected cells (P <0.05), but the luciferase activity of circBIRC6-MUT did not change signi cantly (Fig.3E). Contrasted with the MUM-2B cells treated with NC probe, the expression level of circBIRC6 in the cells treated with miR-503-3p probe was signi cantly increased (P <0.05, Fig.3F). Contrasted with the Ctrl group, the expression level of miR-503-3p was signi cantly increased in the si-circBIRC6 group (P <0.05), and the expression level of miR-503-3p in the pCDS-circBIRC6 group was signi cantly reduced (P <0.05, Fig. 3G).

miR-503-3p targeted ERK and declined the DDP resistance of choroidal melanoma cells
We predicted with TargetScan version 7.2 and ERK was identi ed as a potential target for miR-503-3p (Fig.4A). The luciferase activity of co-transfected cells with miR-503-3p mimic and ERK-WT was signi cantly reduced (P <0.05), but the luciferase activity of ERK-MUT did not change signi cantly (Fig.4B). In addition, it was found that the expression level of miR-503-3p was signi cantly raised in the DDP-sensitive group and DDP-resistant group contrasted with that in the Normal group (P <0.05), and the expression level of miR-503-3p was raised more signi cantly in the DDP-resistant group (P <0.05, Fig.4C).
The expression level of miR-503-3p was negatively correlated with Erk in choroidal melanoma (Fig.4D). Contrasted with the CM group, the mRNA and protein expression levels of Erk in the MUM-2B group and the MUM-2B/DDP group were signi cantly increased (P <0.05), and the mRNA and protein expression levels of Erk was increased more signi cantly in the MUM-2B / DDP group (P <0.05, Fig.4E, 4F). miR-503-3p reduced the DDP resistance of choroidal melanoma cells by ERK As shown in Fig.5A, the expression level of ERK protein in the miR-503-3p mimic group was signi cantly reduced contrasted with that in the Ctrl group (P <0.05), while co-transfection of miR-503-3p mimic with pCDS-ERK reversed the effect of miR-503-3p mimic on the expression level of ERK protein (P <0.05). In addition, miR-503-3p mimic signi cantly inhibited the viability of MUM-2B / DDP cells (P <0.05), while cotransfection of miR-503-3p mimic with pCDS-ERK reversed the effect of miR-503-3p mimic on cell viability (P <0.05, Fig.5B, 5C). Similarly, miR-503-3p mimic effectively reduced IC50 value in DDP-resistant cells and decreased the protein expression levels of P-gp and MRP1 (P<0.05), while co-transfection of miR-503-3p mimic with pCDS-ERK reversed the effect of miR-503-3p mimic on the protein expression levels of P-gp and MRP1 (P <0.05, Fig.5D). These results indicated that miR-503-3p reduced DDP resistance of choroidal melanoma cells through ERK. circBIRC6 regulated ERK expression and DDP resistance in choroidal melanoma via sponging to miR-503-3p As shown in Fig.6A, the expression level of ERK in MUM-2B/DDP cells transfected with si-circBIRC6 was signi cantly reduced contrasted with that in the Ctrl group, and co-transfection of si-circBIRC6 with miR-503-3p inhibitor or pCDS-ERK reversed the effect of si-circBIRC6 on ERK expression level. In addition, si-circBIRC6 signi cantly inhibited MUM-2B / DDP cell viability (P <0.05), while co-transfection of si-circBIRC6 with miR-503-3pinhibitor or pCDS-ERK reversed the effect of si-circBIRC6 on cell viability (P <0.05, Fig.6B and 6C). Similarly, si-circBIRC6 signi cantly reduced the protein expression levels of P-gp and MRP1 (P <0.05), and co-transfection of si-circBIRC6 with miR-503-3pinhibitor or pCDS-ERK reversed the effect of si-circBIRC6 on P-gp and MRP1 protein expression levels (P <0.05, Fig.6D). These results indicated that the circBIRC6 / miR-503-3p / ERK axis was essential for regulating DDP resistance in choroidal melanoma cells.

Discussion
Choroidal melanoma is a severe primary intraocular malignant melanoma that often occurs in adults. Statistical analysis shows that the incidence of intraocular malignant melanoma has continued to increase in recent years, and the incidence rate of developing countries has gradually approached western developed countries [24]. The death rate of malignant melanoma is as high as 50% [25]. Clinical studies have shown that the median survival time of intraocular malignant melanoma such as choroidal melanoma is only 4 to 5 months [26]. Since cisplatin has been used as a chemotherapy drug, it has become the most important drug for cancer treatment [27]. However, about 25% of patients with choroidal melanoma will develop drug resistance within 6 months of platinum chemotherapy [28]. Platinum resistance is an important factor in the recurrence of choroidal melanoma [29]. Therefore, it is particularly important to study the mechanism of platinum resistance to choroidal melanoma and nd an effective treatment for choroidal melanoma.
CircRNA is a type of endogenous RNA that is widely present in cells and has diverse biological functions. With the development of bioinformatics and whole gene sequencing technology, circRNA is gradually discovered by scholars, and its function in cells is gradually being studied and understood [30]. CircRNA has rich biological functions, which can not only regulate the cell cycle but also promote cell selfproliferation [31]. It can not only play the role of oncogenes, but also play the role of tumor suppressor genes. It has a great correlation with the invasion, distant metastasis and prognosis of tumor patients [32]. Studies have shown that certain circRNAs are related to chemotherapy resistance in osteosarcoma, pancreatic cancer, and breast cancer. For example, studies have found that expression of circ_000401 is raised in NSCLC, and circ_0004015 is involved in the progression of NSCLC through miR-1183/PDPK1 signaling, including TKI resistance [33]. However, few researches have been reported on the role of circRNA in choroidal melanoma resistance, and the relationship between circRNA and choroidal melanoma resistance is currently unknown. CircBIRC6 is a recently discovered CircRNA, research has found that it is abnormally expressed in a variety of malignant tumors [34]. For example, it has been found that circBIRC6 is raised in lung cancer tissues and increases cell viability [35]. The occurrence of cancer resistance is related to the abnormality of multiple genes and their products, including multidrug resistance gene 1 (MDR1), lung resistance-related protein (LRP), etc [36]. P-gp is an MDR1 gene expression product and is an ATP-dependent transporter that can transport intracellular chemotherapeutic drugs out of the cell, thereby reducing the e cacy of the drug and leading to the emergence of drug resistance [37]. This study found that pCDS-circBIRC6 inhibited P-gp and MRP1 protein expression, and si-circBIRC6 increased the resistance to adverse reactions of DDP. The above results indicated that the expression of circBIRC6 was closely related to the regulation of DDP resistance in choroidal melanoma. circRNA can be used as a sponge for miRNA, targeting mRNA to promote cell autophagy, which shows that circRNA may also be involved in regulating cell apoptosis [38]. In the process of tumorigenesis and development, miRNA can not only promote tumorigenesis by regulating the expression of oncogenes, but also inhibit tumorigenesis by inhibiting the expression of oncogenes [39]. Many studies have shown that miRNAs play an pivotal role in the occurrence and regulation of cisplatin resistance [40]. For example, miR-218 can up-regulate expression and further increase the sensitivity of esophageal cancer cells to cisplatin [41]. MiR-503-3p is a tumor found in recent years. It is found that miR-503-3p overexpression can induce breast cancer apoptosis [42]. In this study, miR-503-3p was screened as a target gene of circBIRC6. miR-503-3p was reduced in choroidal melanoma tissues and cell lines, and its expression was closely related to the regulation of DDP resistance in choroidal melanoma. It was veri ed that circBIRC6 regulated DDP resistance in choroidal melanoma cells through miR-503-3p.
MAPK / ERK signaling pathway play a key role in the process of signal transfer from cell surface to nucleus, and regulates cell proliferation and differentiation by activating transcription factors in the nucleus [43]. ERK is a very important member of its family, and it is related to cell growth, transformation, drug sensitivity and other biological behaviors [44]. Studies in a variety of tumors suggest that the activity of ERK protein increases in a variety of malignant tumors, cells form a state of high proliferation and low apoptosis, and the defense ability of various biochemical or other stimulations to promote apoptosis is greatly improved [45]. Therefore, it was speculated that this mechanism may lead to tumor cells bypassing cisplatin induced apoptosis and producing cisplatin resistance. In this study, miR-503-3p was selected as Target gene of ERK through the database. Co-transfection of si-circBIRC6 with miR-503-3pinhibitor or pCDS-ERK reversed the effect of si-circBIRC6 on cell viability and protein expression levels of P-gp and MRP1. It demonstrated that circBIRC6 targeted and inhibited miR-503-3p/ERK expression, thereby increasing the DDP sensitivity of choroidal melanoma cells.

Conclusion
In this study, it was focused on circBIRC6 and con rmed the upregulation of circBIRC6 in choroidal melanoma. In addition, circBIRC6 released ERK by acting as a miR-503-3p sponge, and increased DDP resistance in choroidal melanoma cells. This may provide new horizons for the treatment of choroidal melanoma. The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Consent for publication
Not applicable.

Competing interests
The authors declare that they have no competing interests. Authors' contributions WL and AN performed the molecular studies. LJ performed the animal experiments. YC provided experimental technical support and performed the statistical analysis. NX designed the study and helped to draft the manuscript. All authors read and approved the nal manuscript.