Analysis of public databases
Datasets for this study are available in the Gene Expression Omnibus (GEO) database or in the Cancer Genome Atlas (TCGA) database.
Cell lines and cell culture
The Nthy-ori3-1 cell line, representing thyroid follicular epithelial cells, and three PTC cell lines (KTC-1, BCPAP, TPC-1) were procured from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Additionally, human umbilical vein endothelial cells (HUVECs) were procured from American Type Culture Collection (Manassas, VA, USA). The cell culture was maintained in RPMI-1640 medium (Gibco, Grand Island, NY, USA) that contained 1% penicillin/streptomycin and 10% fetal bovine serum (FBS). Subsequently, The cell lines were subjected to incubation conditions of 37°C and 5% CO2 [24].
Cell transfection
The following constructs were procured from RiboBio Co. (Guangzhou, China): pcDNA empty vector, pcDNA-LINC00894, pcDNA-METTL3, pcDNA-YTHDC2, siRNA normal control (SI-NC), and siRNAs targeting LINC00894 (SI-LINC00894). The process of transfection was carried out utilizing either Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) or X-tremeGENE transfection reagent (Thermo Fisher Scientific, USA) following the protocols [25].
RNA extraction, reverse transcription, and real-time PCR
The TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was utilized to isolate Total RNA from the cells following the protocols. Subsequently, 1 μg RNA was subjected to reverse transcription through the Qiagen kit (Qiagen, Valencia, CA, USA) to obtain cDNA. The resulting cDNA was subjected to qRT-PCR utilizing the SYBR Premix Ex Taq Kit (Takara, Otsu, Japan) on the StepOnePlus™ Real-Time PCR System (Applied Biosystems; Shanghai, China). The 2−ΔΔCt method was utilized for quantifying relative expression, with GAPDH serving as the endogenous control [26]. The primer sequences were presented as follows:
LINC00894: forward:5′-CCAAATCTGACACACCATAGC-3′;
reverse:5′-GAACACAGCATGCAGGTAAT-3′.
GAPDH:forward:5′-GCTGTAGCCAAATCGTTGT-3′,
reverse:5′-CCAGGTGGTCTCCTCTGA-3′.
METTL3:forward:5′-AAGCTGCACTTCAGACGAAT-3′,
reverse:5′-GGAATCACCTCCGACACTC-3′.
YTHDC2:forward:5′-CAAAACATGCTGTTAGGAGCCT-3′,
reverse: 5′- CCACTTGTCTTGCTCATTTCCC-3′.
Tube formation of HUVECs
The precooled Matrigel was introduced into the 96-well plate and subjected to incubation at 37°C for 30 min. HUVECs（2 × 104 cells/well）were suspended in 200μL conditioned medium and subsequently transferred to the corresponding well and incubated with supeRNAtants from specified KTC-1 and BCPAP cells in 5%CO2 at 37°C for 8-12 h[27]. The images of tube structures were detected under inverted microscopy at a 100× bright-field microscope, and photographs were recorded. Use the ImageJ software to determine the number of meshes.
Cell counting kit-8 (CCK-8) assay
Cell viability was conducted through CCK-8 [28]. KTC-1 and BCPAP cells were re-suspended and subsequently seeded into 96-well plates with a total of five wells allocated to each group. Every well was filled with a quantity of 2×103 cells and 100μL of the medium, which was then subjected to incubation at 37℃ with 5% CO2 for 0, 1, 2, 3, 4, and 5 consecutive days. The 10 μLCCK-8 solution (KeyGEN, Nanjing, China) was pipetted into a single well in a 96-well plate, followed by a 2-hour incubation period. The cellular proliferation rate was measured at 450 nm absorbance through an enzymatic marker.
Colony formation assay
A colony formation assay was utilized for evaluating the colony formation ability of cells. The PTC cells were inoculated into 6-well plates (300-500 cells /well) and then subjected to the culture at 37°C and 5%CO2 for 14 days, and the replacement of the medium occurred every 3-5 days on a regular basis. Following 14 days, the medium was removed and fixed with paraformaldehyde. Afterward, the cells were subjected to staining with 0.1% crystal violet and were followed by a 20-min incubation at room temperature (RT). Colony counts were photographed [28].
MeRIP-qPCR
The Magna meRIP M6A Kit (Millipore, Germany) was utilized to conduct the meRIP assay for the purpose of determining M6A enrichment following the protocols. The PTC cells were subjected to lysis using a complete RIP lysis buffer, following which the total RNA was extracted. The RNA samples went through immunoprecipitation through the utilization of magnetic beads of either anti-M6A antibody or anti-mouse IgG. The RNAs that were co-precipitated went through a purification process and were subsequently dissolved in RNAse-free water. qRT-PCR was used for analyzing M6A enrichment of binding RNA targets.
Western blotting analysis
The PTC cells were subjected to protein extraction through RIPA buffer (Beyotime Biotechnology, Shanghai, China) containing protease and phosphatase inhibitors (Merck, Germany). After separating the proteins using SDS-PAGE, they were transferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was then blocked for 60 min with QuickBlock™ Western Blocking Buffer (P0252, Beyotime Biotechnology, Shanghai, China) and then subjected to incubation overnight at 4℃. After five times of washing with TBST for 5 min, the secondary antibody combined with horseradish peroxidase (HRP) was observed at RT for one hour, and the protein was detected by BeyoECL Moon (Beyotime Biotechnology, Shanghai, China).
Statistical analysis
Statistical analysis was conducted by SPSS 19.0 software (IBM, Chicago, USA) and GraphPad Prism 8 (San Diego, USA). The study employed a one-way analysis of variance (ANOVA) for determining the significant differences between multiple groups, while the Student's t-test was utilized to evaluate the significant differences between the two groups. The data were reported as mean ± standard deviation (SD). p<0.05 was deemed to be a significant difference.