Animals and animal care
The animal experiments were performed by the Animal Care and Use Committee of Jilin University which in accordance with the Guidelines on Animal Care and Use of Animals in Research (Grant No. SY20191210). Ten nude mice (female, six weeks old) were purchased from the Jilin University. The mice were housed at 24-26 °C and 40% to 60% humidity. All operations were carried out under ARRIVE guidelines (https://arriveguidelines.org).
Cell culture and quercetin treatment
The human uveal melanoma OCM-1, human skin melanoma cells SK-MEL-1 and mouse melanoma B16 cell lines were donated by Changchun University of Chinese Medicine. The cell lines were cultured in DMEM (Gibco, USA) supplemented with 10 % FBS (Gibco) at 37°C and 5% CO2. The cells (2 × 105 cells/mL) were treated with 50 μM quercetin (Sigma-Aldrich, USA) for 48 h.
Overexpression and knockdown of TET1 and miR-17
Synthetic RNA oligonucleotides targeting TET1 were purchased from GenePharma (Shanghai, China). FH-TET1-pEF was purchased from Addgene (Addgene plasmid # 49792). The mimics and inhibitor of miR-17-3p were purchased from GenePharma (Shanghai, China). OCM-1 cells were transfected with si-TET1, FH-TET1-pEF, miR-17-3p mimics, miR-17-3p inhibitor for 48 h. Control (Con) cells were transfected with nonspecific (Nc) siRNA.
Cell growth analysis
The cells were counted with the Cell Counting Kit-8 (CCK-8) assay kit (Meilunbio,Dalian,China) as previously described [13]. The cells (4 × 103) were cultured in 96-well plates and incubated for 48 hours. Then, CCK-8 solution (10 μL) was added to the cells for 2.5 h. The absorbance was measured at 450 nm using a microplate reader (Infinite M200, TECAN).
Cell cycle and apoptosis analysis
To analyze the cell cycle of OCM-1, PI staining was used. In brief, OCM-1 cells (1 × 10 6 cells/mL) were treated with quercetin for 48 h. The cells fixed with 70% ethanol for 2 h at 4 °C. The OCM-1 cells were incubated with PI and RNase A for 30 min. An AccuriTM C6 flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) was used to analyze the cell cycles.
The procedure for cell apoptosis detection has been previously described [14]. Briefly,the OCM-1 cells were stained with Annexin V-FITC/PI after treatment for 48 h. Following incubation, the cells were collected at a concentration of 1×10 6 cells/mL. According to the manufacturer’s instructions, Annexin V-FITC and PI were added for each treated cell sample. The OCM-1 cells were incubated for 30 min. The AccuriTM C6 flow cytometer was used to analyzed.
Cell migration and invasion analysis
Wound healing assay was used to detected cell migration of OCM-1 cells. The OCM-1 (5 × 105) cells were cultured and treatment with quercetin, si-TET1 or FH-TET1-pEF. Then, the scratched line was created with no serum-containing culture medium in these cells. Cell migration was measured using the scratched area at 0 h, 24 h and 48 h.
For cell invasion assays, OCM-1 cells (3 × 104) were cultured 20 μL Matrigel and treatment with quercetin, si-TET1 or FH-TET1-pEF. Next, OCM-1 cells were contained with 10 % FBS for 24 h. Then, 0.2 % crystal violet dye (Solarbio, China) was used to stain cells.
Melanoma models of nude mice
To build animal melanoma models, the left flank areas of nude mice were injected subcutaneously with OCM-1 cells (3 × 10 5 cells). The tumors were observed nude mice after seven or eight days. Then, the nude mice were divided randomly into two groups of five mice each. The treatment group was given 100mg/kg quercetin per day by intraperitoneal injection for 21 days. The Con group was given equal quantities of saline. Animals will be euthanized in advance, if they lose body weight significant significantly, become incapacitated, or have tumors larger than 1.5 cm in diameter during the period. The tumor size was measured. The length (L) and width (W) of tumors were recorded and the calculation of tumor volumes was according to (L × W 2 / 2). The allocation, the outcome assessment and the data analysis were completed with blinding.
Gene expression analysis
Total RNA was isolated from the OCM-1, B16 cells and tumor tissue using TRNzol reagent (TIANGEN, Beijing, China). The isolated RNA samples were first treated with DNase I (Invitrogen, Carlsbad, CA, USA), then reverse transcribed to cDNA using cDNA First Strand Synthesis Kit (TIANGEN, Beijing, China). Quantitative real-time PCR (qPCR) was used to measure gene expression. The sequences of primers are listed in Table S1. qPCR was performed using the BIO-RAD iQ5 Multicolor Real-Time PCR Detection System with the SYBR Green I Real-Time PCR Kit (TIANGEN, Beijing, China). The PCR conditions were 94 °C for 3 min, followed by 35 cycles of denaturation at 94 °C for 10 s, annealing at 60 °C for 15 s, and extension at 72 °C for 30 seconds as described by Teng et al[15]. The 2-ΔΔCT method was used to determine relative gene expression. The reference gene is GAPDH and U6. All experiments were repeated three times for each gene.
Western blot analysis
The proteins of cells and tumors were extracted with RIPA lysis buffer. The BCA protein assay kit (TIANGEN, Beijing, China) was used to detect protein concentrations. 10% SDS-polyacrylamide gels were used to separate proteins. Then, the proteins were transferred to PVDF membranes at 220mA. After blocking with 5% skim milk for 2 hours, the membranes were incubated with primary antibodies overnight at 4 °C. As previously reported[15], the primary antibodies were used including rabbit anti-TET1 (Abcam), anti-TET2 (Abcam, USA), anti-TET3 (Abcam, USA), and mouse anti-GAPDH (Abcam, USA). Subsequently, the membranes were incubated with HRP-conjugated secondary antibodies (anti-mouse or anti-rabbit, Invitrogen, USA) for 1 h. The proteins were detected using ECL Super Signal (Pierce, USA).
Hematoxylin and eosin (H&E) staining
The steps for HE staining are as described in our previous article[16]. Tumor tissues from the Con and quercetin groups were fixed in 4% paraformaldehyde for 48 h. Then, the samples embedded in paraffin wax and cut into 5 μm sections. The slides were stained with hematoxylin and eosin (H&E) and cancer cell infiltration was determined by observation under a light microscope.
Immunofluorescence (IF) staining
The 4% paraformaldehyde solution was used to fix OCM-1 cells for 15 min. Then, the OCM-1 cells were contained with 0.5% Triton X-100 for 30 min. The 5hmC antibody (1:400; # 51660S, Cell Signaling Technology, USA) were performed. The OCM-1 cells were incubated with anti-mouse antibody (1:400, BA1031, BOSTER, USA). The cells used 4,6-diamidino-2-phenylindole (DAPI, Meilunbio, China) with an antifade mounting medium and imaged using fluorescence microscope.
Statistical analyses
The data of qPCR and flow cytometry (FCM) were analyzed by Student’s t-tests using GraphPad Prism 5.0 (GraphPad Software, Inc., San Diego, CA). A p-value of < 0.05 was considered statistically significant.