Research Paper Volume 12, Issue 24 pp 24967—24982

Figure 4. Silencing of METTL3 reduces MYC mRNA m6A modification and impedes the MYC expression pattern in GC cells. (A) m6A modification levels in GC tissues (n = 45) and GC cells were examined by dot blot assays, * p < 0.05 vs. the paracancerous tissues or GES-1 cells. (B) METTL3 and MYC co-expression in GC from the TCGA database. Each point represents a sample, the abscissa indicates the METTL3 expression pattern, the ordinate indicates the MYC expression pattern, and the upper left indicates the correlation coefficient and p value. (C and D), MYC mRNA expression and protein expression patterns in GC and paracancerous tissues (n = 45) were determined by RT-qPCR (C) and Western blot assay (D), normalized to GAPDH, which were expressed as mean ± standard deviation, and test with paired t-test, * p < 0.05 vs. the paracancerous tissues. (E and F) MYC mRNA expression and protein expression patterns in AGS and MKN-45 cells were determined by RT-qPCR (E) and Western blot assay (F), normalized to GAPDH. (G) Representative Western blots of MYC protein and its quantitation in AGS and MKN-45 cells after silencing METTL3, normalized to GAPDH. (H) Me-RIP and RT-qPCR were used to examine m6A levels of MYC mRNA in AGS and MKN-45 cells after silencing METTL3. (I) The binding of METTL3 to MYC mRNA assessed by PAR-CLIP assay in GC cells following METTL3 silencing. The above results were measurement data, and expressed as mean ± standard deviation. Data in panels A (the left), (C and D) were compared by paired t test, and in panels A (the middle and the right) and (E–I) by one-way ANOVA with Tukey’s post hoc test. The cell experiment was repeated 3 times independently.