Specimen preparation and data collection
Tissue samples (both CM and normal choroidal tissues) were collected from 36 volunteer patients admitted to our hospital during October 2016 to December 2021. CM tissues were resected from patients with clinically and pathologically diagnosed CM, while normal choroidal tissues were excised from patients with mechanical ocular trauma or ocular atrophy. All participants provided written informed consent and ethical approval was issued by the ethics committee of The First Hospital of China Medical University. The samples were divided into two portions, one was soaked in formalin immediately, and the other kept at -80°C.
Western blot (WB) assay
Total protein from tissues or cell lysates was extracted using a buffer containing 1% phenylmethylsulfonyl fluoride (PMSF), and protein concentrations were determined using a bicinchoninic acid (BCA) assay kit (Beyotime Institute of Biotechnology). Protein (40 μg/lane) was denatured and isolated by SDS (10%)-polyacrylamide (PAL) electrophoresis (140 V, 50 min). The protein was transferred to a polyvinyl formal (PVF) membrane (350 mA, 90 min) blocked with 5% milk (0% fat), which was then sealed in a container at 37 °C for 60 min. The membrane was subsequently incubated with 5% milk (0% fat) containing the following primary antibodies at 4 °C for 12 h: anti-METTL14 (1:1,000, 51104S, Cell Signaling Technology, Danvers, MA, USA), anti-runt-related transcription factor 2 (RUNX2, 1:1,000 , 12556S, Cell Signaling Technology, Danvers, MA, USA), anti-β-catenin (1:1,000, 8480S, Cell Signaling Technology, Danvers, MA, USA), anti-phospho-GSK3β (1:1,000, 9322S, Cell Signaling Technology, Danvers, MA, USA), anti-GSK3β (1:1,000, 9315S, Cell Signaling Technology, Danvers, MA, USA), and anti-β-actin (1:1,000, 3700S, Cell Signaling Technology, Danvers, MA, USA). Membranes were rinsed three times using Tween-20 buffer for 5 min each and incubated using the corresponding secondary antibodies at 37 °C for 1 h. Density was measured using ImageJ software (National Institutes of Health, Bethesda, MD, USA) and protein bands were normalized to β-actin.
Sections of CM and normal tissue samples were embedded in paraffin, followed by deparaffinization and rehydration. The slides were incubated overnight at 4 °C with the primary antibodies anti-METTL14 (1:1,000, HPA038002, Sigma-Aldrich, St. Louis, MO, USA) and anti-RUNX2 (1:1,000, MAB2006, RD, USA), followed by addition of secondary antibodies to an avidin-biotin-peroxidase complex (e.g., biotinylated anti-rabbit and anti-mouse antibodies in goats). The slides were kept at 25 °C for 60 min and successively stained with DAB reagent and counterstained with hematoxylin. Images were captured using an inverted microscope (EVOS XL system, AMEX1200; Life Technologies Corp, Bothell, WA, USA) (magnifications = 200× and 40×).
Cell lines and cell culture
The human CM cell lines OCM1 and MUM-2B were purchased from the Chinese Academy of Sciences Type Culture Collection Cell Bank (Shanghai, China). The passage time was ≤180 d with mycoplasma elimination performed by the company. Using six-well plates, we cultured OCM1 cells in RPMI medium (Hyclone; GE Healthcare) and 10% FBS, and MUM-2B cells were allowed to proliferate in DMEM medium (Hyclone; GE Healthcare) and 10% FBS. All CM cell lines were maintained at 37 °C in 5% CO2. At a cell fusion rate of 90%, cells were incubated with trypsin (1 mL) for 5 min and neutralized with growth medium (1 mL). Finally, they were centrifuged and passaged.
At a confluence of 60%, the OCM1 and MUM-2B cells were transfected with METTL14 siRNA (JTS scientific, Wuhan, China) using Lipofectamine 3000 (Invitrogen, Waltham, MA, USA). siRNA sequences were as follows (5′-3′): siMETTL14-1# (sense GGAUGAAGGAGAGACAGAUTT and anti-sense CCUGGGAAGACUAAGACUUTT), siMETTL14-2# (sense CAAAGAUGAGCAGAGAGAAAUUGCU and anti-sense AGCAAUUUCUCUCUGCUCAUCUUUG).
RNA extraction and qRT-PCR
Extraction of total RNA from OCM1 and MUM-2B cells was conducted 2 d after siRNA transfection using RNAiso Plus (Takara Biotechnology, Dalian, China). cDNA was generated using Prime Script RT Master Mix (Takara Biotechnology, Dalian, China), while the SYBR Premix ExTaq™ kit (Takara Biotechnology, Dalian, China) was employed for RT-PCR. A LightCycler 480 II system (Roche, Basel, Switzerland) was used for RNA detection with β-actin as the internal control. The primers for METTL14, RUNX2, and β-actin were as follows (5′-3′): METTL14 (sense GAACACAGAGCTTAAATCCCCA and anti-sense TGTCAGCTAAACCTACATCCCTG), RUNX2 (sense GCGCATTCCTCATCCCAGTA and anti-sense GGCTCAGGTAGGAGGGGTAA), and β-actin (sense CATGTACGTTGCTATCCAGGC and anti-sense CTCCTTAATGTCACGCACGAT). The 2-ΔΔCt method was used to determine the relative RNA expression levels.
Transfected cells (1 × 106) in growth medium (200 μL, containing no serum) were transferred to the upper chamber of a transwell plate and another 600 μL of the medium was added to the bottom chamber. Cells were incubated at 37 °C in 5% CO2 for 1 d. Using a swab, we removed residual cells from the upper chamber and fixed the cells by incubation with 4% paraformaldehyde for 600 s. Next, a crystal violet stain was added, and the cells were further incubated for 10 min. Images were collected using an optical microscope and analyzed using ImageJ (National Institutes of Health, Bethesda, MD, USA). Additionally, the transwell filter was coated with Matrigel (BD, San Diego, CA, USA) and dried overnight for invasion assays.
METTL14 and RUNX2 knockdown and overexpression plasmids were purchased from GeneChem (Shanghai, China). All the procedures were performed in accordance with manufacturer's instructions. Selection of stable transformants was conducted over two weeks using puromycin (5 μg/mL) and confirmed by WB assay for METTL14 and RUNX2. Cells with confirmed knockdown and overexpression were stored for further tests.
Female BALB/c nude mice (n = 15) aged 4-6 weeks were provided by a commercial contractor and housed in a pathogen-free environment.
In the analysis of METTL14-mediated metastasis, we injected 150 μL buffer containing 1.0×105 MUM-2B cells transfected with an empty vector (n = 5) and shMETTL14 (n = 5), respectively, via the lateral tail vein. After 45 d, we excised the lungs to count the metastatic tumors and observed histological sections using hematoxylin and eosin staining.
In the analysis of RUNX2-mediated metastasis, injection of 1.0×105 MUM-2B cells (transfected with empty vector and RUNX2-knockdown, respectively) in buffer (150 μL) was achieved through the lateral tail vein. 45 days later, lung separation was executed to count the metastatic tumors. All experimental procedures were approved by the ethics committee of The First Hospital of China Medical University (CMU2021090).
Characterization by PET/CT imaging
Tumor-bearing mice (n=5 per group), identified using METIS PET, were injected with ~100 μCi [18F] fluoro-D-glucose via the tail vein. Anesthesia was conducted using isoflurane (3% and 2% for induction and maintenance, respectively, in pure O2) and mice were placed in a prone position in the center of the scanner. The obtained PET and CT metadata were reconstructed and each image was statistically analyzed. An organ shape was contoured on each slide, which comprises a part of this organ in the fused PET-CT image.
The Cancer Genome Atlas (TCGA) data
We conducted a bioinformatic analysis using data from the TCGA database. Survival curves for CM patients with different levels of RUNX2 were selected using a gene expression profiling interactive analysis (GEPIA) (http://gepia.cancer-pku.cn/) based on a suitable expression threshold.
Methylated RNA immunoprecipitation (MeRIP)
We evaluated the abundance of specific mRNA transcripts in m6A immunoprecipitation and input groups by qPCR using a MeRIP kit (BersinBio, Guangzhou, China). RNA was randomly divided into 100 nucleotide fragments and immunoprecipitation was performed with anti-m6A or anti-rabbit IgG antibodies linked by A/G magnetic beads. A magnetic frame was used to elute the m6A-precipitated RNA. Enriched RNA was extracted by phenol-chloroform and ethanol precipitation, and m6A modification towards particular genes was determined by qPCR analysis, using the primer for MeRIP-qPCR RUNX2-MeRIP-3′ UTR: TCCTCTGAAAAGGCAGCAGG; GCATGCCACAGAAGGACTCT). Note that the m6A sites of specific genes were obtained online (http://www.cuilab.cn/sramp).
Plasmid development and dual-luciferase reporter assay
Wild-type/mutated pmiR-RB-Report-RUNX2-3′-UTR plasmids (GeneChem, Shanghai, China) were transfected with METTL14-OE and vector MUM-2B cells, respectively. The experimental groups were divided into a RUNX2-WT+vector, RUNX2-WT+METTL14-OE, RUNX2-Mut+vector, and RUNX2-Mut+METTL14-OE group.
All tests were executed in triplicates, and all data were described as mean ± standard deviation (SD). Software GraphPad Prism of version 8.0 (La Jolla, CA, USA) was employed for statistical analysis. Inter-group differences were assessed using a Student’s t-test. The differences in expression levels between paired samples were assessed using a Wilcoxon signed-rank test. Survival rates were estimated using the Kaplan–Meier log-rank method.