Website UALCAN (http://ualcan.path.uab.edu/) was used to visually obtain gene expressions in different sample types (ccRCC tissues and normal kidney tissues), different pathological grades and clinical stages of ccRCC with expression data provided by The Cancer Genome Atlas (TCGA) database. METTL13 expressions in ccRCC tissues and adjacent normal tissues were also acquired from a dataset (GSE53757) of the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo/) . Website GEPIA (http://gepia.cancer-pku.cn/) directly produced survival curves of ccRCC patients with high and low METTL13 expression levels based on an appropriate expression threshold. Transcriptome data of kidney renal clear cell carcinoma (KIRC) cohort was downloaded from TCGA database and differentially expressed genes (DEGs) was filtered out with |logFC|>1 and false discovery rate (FDR) < 0.05 as the criteria. We performed weighted gene co-expression network analysis (WGCNA) with the WGCNA package in R (The WGCNA package in R). ClusterProfiler R package was used to determine gene modules’ gene ontology and KEGG pathway enrichments regarding FDR < 0.05 as threshold.
All the ccRCC tissues and their corresponding adjacent normal tissues were obtained from the urology surgery department of the first hospital of China Medical University (Shenyang, China). This study was approved by Research Ethics Committee of China Medical University and all the patients had supplied the written informed consent.
Cell lines and cell culture
Human cell lines, 786-O, 769-P, OS-RC-2, Caki-1 and ACHN were purchased from Chinese Academy of Sciences Type Culture Collection Cell Bank (Shanghai, China). 786-O, 769-P and OS-RC-2 cells were cultured in RPMI medium (Hyclone; GE Healthcare), while Caki-1 cells and ACHN cells were treated with McCoy's 5A medium Hyclone; GE Healthcare) and MEM medium (Hyclone; GE Healthcare), respectively. All the mediums contained 10% fetal bovine serum (FBS; Biological Industries, Beit-HaEmek, Israel) and cells were cultured in an atmosphere of 37°C along with 5% CO2.
Two strands of siRNAs targeting at METTL13 were designed and purchased from JTSBIO Co. (China), sequences of which were as following: si-METTL13#1 (sense: GCGGGGUGCUACAUAAAUATT; anti-sense: UAUUUAUGUAGCACCCCGCTT), si-METTL13#2 (sense: GCUCUGCCCUUCAGAUCUUTT; anti-sense: AAGAUCUGAAGGGCAGAGCTT). Usage of LipofectamineTM3000 (Invitrogen, USA) was involved in siRNA transfection with the protocol provided by manufacturer's guide- lines. METTL13 overexpression plasmid was purchased from GeneChem (Shanghai, China) and transfection was performed according to the manufacture’s guidance.
Quantitative real-time PCR (qRT-PCR)
Total RNA was extracted from tissue samples and cells with the use of RNAiso Plus (Takara Biotechnology, Dalian, China) under the guidance of manufacture’s recommendations, following which Prime Script RT Master Mix (Takara Biotechnology, Dalian, China) was utilized to conduct reverse transcription to synthesize cDNA. With the use of a SYBER Premix Ex TaqTMKit (Takara Biotechnology, Dalian, China), qRT-PCR was conducted by LightCyclerTM 480 II system (Roche, Basel, Switzerland), after which the 2-ΔΔCt method was used to calculate the relative expression level of each sample referring to internal β‐actin or GAPDH expression. Information of primer sequences is listed in Supplementary table 1.
Western blotting assay
Total protein from patient samples or cells was obtained by RIPA lysis buffer with 1% Phenylmethylsulfonyl fluoride (PMSF) contained and protein concentrations were detected by bicinchoninic acid (BCA) assay kit (Beyotime Institute of Biotechnology). Equal mass of proteins (30 μg/lane) were used for electrophoresis in 10% SDS- polyacrylamide, followed by PVDF membrane (0.2 μm) transfer, membrane block by 5% non-fat milk, primary and secondary antibody incubation as well as image capture by an EasySee Western Blot kit (Beijing Transgen Biotech, Beijing, China) and a chemiluminescence system (Bio-Rad, CA, USA). Information of primary antibodies is listed in Supplementary table 2.
5-Ethynyl-2′-deoxyuridine (EdU) assay
EdU assay was performed with the usage of an EdU kit (BeyoClickTM, EDU-488, China). Cells were co-cultured with EdU working solution (1:1,000) in the atmosphere of 37°C and 5% CO2 for 2~4 hours, followed by fixation with 4% paraformaldehyde for 30 min and treatment with 0.3% Triton X-100 for 30 min at room temperature. Then, according to the manufacture’s protocol, cells were co-incubated with click reaction solution for 30 min at room temperature in a dark environment, after which cells were treated with Hoechst solution for 10 min. We used a fluorescence microscope (Olympus Corporation, Japan) to capture images with a magnification of 400 ×and cell counting was conducted by ImageJ software.
Cell viability assay (CCK-8 assay)
Counting Kit-8 (CCK-8) solution (Bimake, USA) was added to each well of 96-well plates to the concentration of 0.5 mg/ml, where 2.0 × 103 cells had been initially seeded. After Incubation for one hour at 37°C with 5% CO2, absorbance at 450 nm was measured by plate reader (Model 680; Bio-Rad Laboratories).
When the density of cells in 6-well plates reached above 90%, we used a 1-mL pipette tip to vertically scratch an artificial wound in the middle of the wells. Then cells were washed with PBS and new FBS-free medium was added. Images were obtained with the help of an inverted microscope (EVOS XL system, AMEX1200; Life Technologies Corp, Bothell, WA, USA) under a magnification of 100 ×. After cultured for 48 h, cell images were re-obtained.
Cell migration and invasion assay
8-μm-pore transwell chambers in 24-well plates (Corning Costar, Corning, NY, USA) were used. Chambers coated with Matrigel (BD, San Diego, CA, USA) were for cell invasion detecting, while those without Matrigel coating were used to determine cell migration. 600μl 10%-FBS containing medium was placed into each bottom chamber, while equal number of suspended cells (1.0 ~ 1.5 × 104 cells for migration assay, 3.0 ~ 4.0 × 104 cells for invasion assay) in 200μl medium without FBS were imbedded onto each upper chamber. After incubated at temperature of 37°C along with 5% CO2 for certain time (24h for 786-O, Caki-1, OS-RC-2 and 48h for ACHN)，suspended cells in the upper chamber were washed out, while cells adhering to the bottom membrane were stained by crystal violet. Images were gained by using the inverted microscope described previously under a magnification of 200 × and cell counting was dependent on software ImageJ.
Co-immunoprecipitation (Co-IP) assay
Cells were lysed by RIPA lysis buffer containing 1% PMSF and 1% protease inhibitor. A certain amount of cell lysate was isolated as input, while 5μg primary antibody (METTL13, abcam, ab186002; c-Myc, Santa Cruz, sc-40) or homologous IgG (Santa Cruz Biotechnology) was co-incubated with remaining lysate at 4°C overnight. Then, 30-μl protein A/G-beads was co-incubated with the lysis solution at 4°C for an hour, after which beads was extracted and washed by washing buffer three times. Next, protein was isolated from beads into 2 × protein loading buffer after co-incubation at 100°C for 15min. Western blot was finally conducted.
Tissues previously formalin-fixed and paraffin-embedded were sliced into 4-μm slides. Following procedures were as previously described , which involved use of rabbit anti-METTL13 antibody (GTX120626, GeneTex, USA) and an UltraSensitiveTM SP (Mouse/Rabbit) IHC kit (Maxin-Bio, Fuzhou, Fujian, China) according to the manufacture’s guidance. Images were captured by the inverted microscope with magnifications of 200 × and 400 ×.
Experiments with animals involved were approved by China Medical University Ethics Committee of Medical Experimental Animal Welfare and were conducted following the institute’s guidelines. Female BALB/c-nude mice of 4-6 weeks old, purchased from Beijing Vital River Experimental Animal Technology Co. Ltd., were housed in a pathogen-free environment at Experimental Animal Department of China Medical University. As for the tumorigenicity study, 1 ×106 OS-RC-2 cells (empty vector or METTL13 overexpression) in 150μl serum-free 1640 medium containing 40% Matrigel were injected subcutaneously into flank of each mouse, 30 days after which mice were euthanized and tumors were excised. Primary tumors were measured for their size and weight. and as for the metastasis study, 1 ×105 OS-RC-2 cells (empty vector or METTL13 overexpression) in 150μl pathogen-free PBS were injected into per mouse via its lateral tail vein. After 45 days, lungs were seperated and metastatic tumors were counted. HE staining was involved in observing serial histological sections of the lungs.
Each experiment was performed independently for at least 3 times and data were expressed as the mean ± standard deviation (SD). Software GraphPad Prism of version 8.0 (La Jolla, CA, USA) was used perform all the statistical analysis, which involved Mann-Whitney U-test and Student's t test. As for all the data, P < 0.05 was regarded as statistically significant. * indicates P < 0.05; ** indicates P < 0.01, *** indicates P < 0.001.