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. 2021 Aug 26;20:108. doi: 10.1186/s12943-021-01403-w

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© The Author(s) 2021

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 4 — DDX56 post-transcriptionally regulates expression of the Wnt signaling pathway-related genes. a GO (KEGG) enrichment analysis of down-regulated mRNAs (> twofold) in a mRNA microarray experiment revealed that the KEGG terms “pathways in cancer” and “Wnt signaling pathway” were significantly enriched in down-regulated mRNAs in DDX56 siRNA- vs. control siRNA-transfected cells. b Heatmap showing WNT signaling-related genes differentially expressed (> twofold) between scramble siRNA (left) and DDX56 siRNA-treated (right) H226 cells in a mRNA microarray experiment. c qRT-PCR analysis of CTNNB1, WNT2B, WNT5A, FZD7, FZD6, SMAD1, and SMAD2 mRNA expression levels in DDX56-specific siRNA (siDDX56-mix) or negative control siRNA- (siNC) transfected H226 cells showed that DDX56 reduction significantly reduced mRNA expression of Wnt pathway-related genes CTNNB1, WNT2B, FZD7, FZD6, SMAD1, and SMAD2 except for WNT5A in H226 cells. d and e Western blot analysis of β-catenin, Wnt2b, and DDX56 protein expression level in H226 and SK-MES-1 cells transfected with DDX56-specific siRNAs or control siRNA, confirming reduced protein expression of β-catenin and Wnt2b following DDX56 reduction in H226 (d) and SK-MES-1 (e) cells. f and g Western blot analysis of the expression levels of β-catenin, Wnt2b and FLAG tag-fused DDX56 protein in H226 and SK-MES-1 cells transfected with pCMV-DDX56 or control pCMV vectors, confirming enhanced protein expression of β-catenin and Wnt2b following DDX56 overexpression in H226 (f) and SK-MES-1 (g) cells. GAPDH expression was used as a loading control. h Nascent transcript analysis showed no significant variation in the newly synthesized EtU-labeled RNA of CTNNB1 and WNT2B, whereas a significant decrease in the total (steady-state) RNA levels of CTNNB1 and WNT2B was observed after siRNA-mediated DDX56 knockdown in H226 cells. Gene expression levels were measured by qRT-PCR and standardized with GAPDH. Data are graphed as mean ± SD (n = 4). * P < 0.05