Tissue sample
The cancerous tissue and adjacent non-cancerous tissues of 40 patients who underwent primary choroidal melanoma surgery were collected from the the Second Clinical Medical College of Jinan University, Shenzhen People's Hospital. All patients received DDP chemotherapy in The Second Clinical Medical College of Jinan University, Shenzhen People's Hospital. The specific chemotherapy regimen was as follows. Patients were treated with intravenous infusion of 75mg /m 2 DDP, repeated every 21 days for a maximum of six courses. According to the literature, classification of patients' DDP sensitivity or resistance was performed. Patients resistant to DDP did not undergo surgery and all patients signed informed consent.
Cell culture and transfection
Normal human choroid melanocytes (CM), choroid melanoma cell line (MUM-2B) and MUM-2B / DDP cells were purchased from Shanghai Fudan (IBS) Cell Resource Center. MUM-2B was cultured in RPMI1640 medium containing 10% fetal bovine serum. MUM-2B/DDP was stably cultured in RPMI1640 medium with cisplatin concentration of 0.1 mg/L and 10% FBS. Both MUM-2B and MUM-2B/DDP were cultured at 37 °C with 5% CO2 saturation humidity. The cells in logarithmic growth stage were transfected and the fusion rate reached 60%. The transfection operation was strictly performed in accordance with Lip2000 and gene fragment instructions. The experiments were divided into blank groups, NC group, and transfection group. CircBIRC6, miR-503-3p mimics and miRNA-590-5p inhibitors were designed and synthesized by RiboBio (Guangzhou, China). To construct circBIRC6, ERK and NF-kb overexpression plasmids, their sequences were cloned into the pCDS-At cloning vector (BioVector, Beijing, China). Lipofectamine 2000 (Invitrogen, Carlsbad, CA) was used to transfect oligonucleotides to MUM-2B and MUM-2B / DDP in the cell.
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 Scientific, Inc.). SYBR-Green (Takara Biotechnology, Co., Lt. (Dalian, China) was used in by qRT-PCR. Amplification 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:
circBIRC6: forward 5′- CTAATTCACCTGGCGGGAG-3′;
circBIRC6: reverse 5′- CGCTTGGATTCCAAGGG-3′
miR-503-3p: forward: 5′- CTGAATGTGAAGGGAATGT-3′,
miR-503-3p: reverse: 5′-GTTCTTCCACATCGGGGCCG-3′
GAPDH: forward: 5′-CGAGAGAATCCGCGGACAT-3′
GAPDH: reverse: 5′-TTGTGCAATACAGCGTGGAC-3′
U6: forward: 5′-GACAGATTCGGTCTGTGGCAC-3′
U6: reverse: 5′-GATTACCCGTCGGCCATCGATC-3′
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% CO2 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 final 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.
Western blot
MUM-2B and MUM-2B / DDP cells in the blank group, NC group, and transfection group were collected after transfection. The supernatant was discarded after centrifugation, lysate was added. After 30 min, it was freezed at -20℃ overnight. The supernatant was centrifuged at 4℃. After protein quantification, PAGE-SDS gel was used for electrophoresis separation of protein samples for 1.5 h. After transferred to PVDF blotting membrane for 1.5 h, it was sealed in TPST containing 5% skimmed milk for 4 h. Then, it was incubated with P-gp (abcam, 1: 1000, UK), MRP1 (1: 1000, CST, US), SMAD3 (1: 1000, CST, US), ERK (1: 1000, CST, US), and anti-GAPDH antibody (1: 1000, Abcam, Cambridge, UK) overnight at 4 ° C. After that, anti-rabbit secondary antibody (1: 1000, Cell Signaling Technology, Boston, MA, USA) was added for 1 h of incubation [23].
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 specific biotin-labeled 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 streptavidin-labeled 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.
Luciferase reporter assay
Wild-type or mutant circBIRC6 was subcloned it into the pm-irGLO vector. PmirGLO, pmirGLO-circBIRC6 or pmirGLO-circBIRC6-mut was co-transfected into cells with miR-503-3p mimic by Lipofectamine 2000 (Invitrogen). After 48 h, luciferase activity was measured using a dual luciferase assay kit (Promega). After centrifugation, luciferase activity was measured by a Modulus TD20 / 20 luminometer (Turner Biosystems, CA).
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 one-way ANOVA. LSD test was used for subsequent analysis. P < 0.05 indicated the difference was significant.