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. 2021 Aug 4;19(3):343–357. doi: 10.20892/j.issn.2095-3941.2020.0661

Figure 1.

Figure 1

The m6A modification writers were highly expressed in CCAs and regulated by IL-6/STAT3 inflammatory signaling. (A) The m6A writers were more highly expressed in cancerous tissues than those in matched adjacent tissues of 38 paired CCA patient samples. The Wilcoxon matched-pairs signed rank test was used to calculate the significance. (B) The m6A writers were more highly expressed in extra-hepatic (ECC) cancerous tissues (N = 20) than in intra-hepatic (ICC) cancerous tissues (N = 18) from CCA patients. The m6A writers were also highly expressed in ICC and ECC samples compared to normal tissues. BB indicates CCA adjacent tissue, and BC indicates CCA cancerous tissue. (C) Dot blots showing the global m6A levels in matched pairs of cancerous and adjacent tissues from patients with CCA. MB indicates methylene blue, which shows the total RNA level. (D) Dot blots showing the global m6A levels between CCA cells treated with or without 20 ng/mL interleukin-6 (IL-6) for 2 h. MB indicates methylene blue. (E) Western blot analysis showed that the expression levels of phosphorylated STAT3 were significantly upregulated in CCA cells treated with 20 ng/mL IL-6 for 2 h. (F) The expression levels of METTL3, METTL14, and WTAP measured by qRT-PCR, and immunoblots of CCA cells treated with 20 ng/mL IL-6 for 2 h. Error bars denote ± SEM (***P < 0.001) in 3 independent experiments. (G) STAT3 was located at the genetic locus of METTL3 and METTL14 in the GES31477 data set. (H) The anti-STAT3 chromatin immunoprecipitation (ChIP) assay showed the enrichment of STAT3 located at the METTL3 and METTL14 gene loci in CCA cells treated with or without 20 ng/mL IL-6 for 2 h. Western blot for the STAT3 of ChIP/IP; glyceraldehyde 3-phosphate dehydrogenase served as the negative control. Error bars denote ± SEM (**P < 0.01) based on 3 independent experiments. (I) Western blot showed the expressions of METTL3 and METTL14 in the sh-METTL3 and sh-METTL14 CCA cells treated with or without 20 ng/mL IL-6 for 2 h, respectively.