Fig. 1

mRNA and protein levels of METTL14 in EC were significantly down-regulated A: METTL14 mRNA expression was measured in tumor and normal tissues based on 70 pairs of EC samples. B, C: Representative plots and grayscale analysis of western blot analysis of METTL14 (calculated by the log2 ratio of EC and matched adjacent tissue, normalized to GAPDH) of 10 pairs of EC samples. ***: P<0.001. T: Tumor tissue; P: Paracancerous tissue."

Tab. 1

Association between METTL14 expression and clinicopathologic parameters"

Fig. 2

METTL14 inhibited EC invasion and metastasis A: Western blot validation of METTL14 silencing and overexpression in AN3CA and KLE cells. B, C: EdU assay was used to determine the effects of METTL14 silencing and overexpression on the proliferation of AN3CA and KLE cells, respectively. D, E: Transwell analysis of the effects of METTL14 silencing and overexpression on the migration and invasion of AN3CA and KLE cells, respectively. F, G: Immunofluorescence imaging detected the cytoskeletal changes of AN3CA and KLE by METTL14 silencing and overexpression, respectively. Phalloidin (red) was used for cytoskeleton staining and DAPI (blue) was used to label nuclei (scale bar=30 μm). H: Representative in vivo images of mice were taken by quantifying luciferase activity in lung regions. I: Metastatic tumor foci in the lungs were photographed and quantified. J: The presence of metastatic tumor lesions was confirmed by H-E staining (scale bar=100 μm). **: P<0.01, compared with Vector group or si-NC group; ***: P<0.001, compared with Vector group or si-NC group."

Fig. 3

ERα identified as a candidate target for METTL14 A: Overall m6A levels of RNA extracted from METTL14 knockdown or overexpressing EC cells were measured by m6A dot blot analysis. Methylene blue staining (left) was used to detect input RNA, and the intensity of dot immunoblotting (right) represents the level of m6A modification. B: Star chart showing the distribution of genes with differential (high or low) m6A peaks (Y axis; fold change >1.5 or <2/3, P<0.05) and differential (up or down) expression (X axis; fold change). Blue dots highlighted with circles represent down-regulated transcripts with reduced m6A abundance upon METTL14 overexpression, which were selected for the following studies. C: The results of MeRIP-seq (blue circles) and RNA-seq (brown circles) were combined using a Venn diagram. The overlap contains 60 transcripts affected by METTL14 for m6A content and expression. The top 10 differentially expressed genes shown in the heatmap (red for up-regulation, blue for down-regulation). D: RNA levels of ERα were detected in METTL14 silenced or overexpressed cells using RTFQ-PCR. E: The effects of METTL14 silencing and overexpression on the protein levels of ERα in AN3CA and KLE cells were detected by Western blot, respectively."

Fig. 4

METTL14 destabilized ERα by regulating m6A modification A: IGV plotted m6A abundance on ERα mRNA in negative control or METTL14-overexpressing KLE cells. Green and pink represent the m6A signal of the input sample, while red and blue represent the signal of the IP sample. Black blocks indicate sites where m6A levels differ between the two groups, with the most significant locations highlighted with grey panes. B, C: Relative enrichment of ERα mRNA associated with METTL14 protein was identified by RIP assay using anti-IgG and anti-METTL14 antibodies. The IgG group was a negative control to exclude nonspecific binding. The Y-axis represents the percentage of input for each IP sample. **: P<0.01, compared with the IgG group. D, E: Detection of m6A modification of ERα by MeRIP-RTFQ-PCR analysis using anti-IgG and anti-m6A antibodies. *: P<0.05, compared with si-NC-m6A group or Vector-m6A group; ***: P <0.001, compared with si-NC-m6A group or Vector-m6A group. F, G: Relative luciferase activity of AN3CA and KLE cells transfected with ERα-wild-type or mutant constructs. ##: P<0.01, compared with Vector group or si-NC group; ###: P<0.001, compared with Vector group or si-NC group."

Fig. 5

ERα knockdown reversed the effect of METTL14 on EC cell proliferation or migration A: EdU assayed the effect of METTL14 silencing and ERα silencing on the proliferation of AN3CA cells. B: Transwell analysis of the effects of METTL14 silencing and ERα silencing on the migration and invasion of AN3CA cells. **: P<0.01, compared with si-METTL14+si-NC group; ***: P <0.001, compared with si-METTL14+si-NC group."