Fig 2: RNA expression profiling identifies MIST1 as a DDX56-downstream target in the regulation of the PTEN-AKT axis. (A) A volcano plot constructed using P-values and fold-change to illustrate the differential downstream target genes between DDX56-silenced MHCC97H cells and control cells (|fold change| =2, P < 0.05). Red dots represent significantly upregulated genes while blue dots represent significantly downregulated genes. (B) The top 20 differential downstream genes from RNA-seq results were validated by RT-PCR in MHCC97H cells. (C-D) The mRNA and protein levels of MIST1 in both MHCC97H and Huh-7 cells were detected using RT-PCR and western blotting, respectively. (E) IHC staining shows the expression level of MIST1 in the subcutaneous tumor. Scale bars: 250 µm (top), 50 µm (bottom). (F) Representative images of DDX56 and MIST1 immunohistochemical staining in human HCC tissues (left). Correlation analysis between DDX56 and MIST1 protein expression in HCC tissues (right). Scale bars: 250 µm (low magnification), 50 µm (high magnification). (G) Effect of MIST1 knockdown on MHCC97H and Huh-7 cell proliferation was determined by colony formation assay. (H) The indicated protein was determined using a western blot analysis of MHCC97H and Huh-7 cells infected with MIST1-silencing lentivirus (shMIST1) or lentivirus control (shCont). (I) Western blot analysis of the PTEN-AKT axis in MIST1-overexpressed HCC cells, showing that PTEN overexpression reversed the effect of MIST1 on the PTEN-AKT axis. Cells were transfected with 3 µg of the indicated plasmids for 72 h.

Fig 3: DDX56 facilitates HCC growth in a MIST1-dependent manner. (A-B) Colony formation (A) and EdU assays (B) revealed that ectopic MIST1 expression in DDX56-silenced cells partially reversed the inhibitory effect on HCC proliferation. Scale bars: 100 µm. (C) Western blot analysis of the PTEN-AKT axis in DDX56-silenced HCC cells showing that MIST1 overexpression reversed the effect of DDX56 on the PTEN-AKT axis. (D-E) DDX56 knockdown decreased liver tumor formation capacity of HCC cells in the orthotopic HCC implantation models, and the effect was reversed by simultaneously overexpressing MIST1. (D) Representative images showing mouse livers with tumor lesions. Red circles indicate the primary liver tumor. (E) Representative images of IHC staining of DDX56 and MIST1 in paraffin-embedded sections obtained from mouse orthotopic HCC models. Scale bars: 250 µm (low magnification), 50 µm (high magnification).

Fig 4: ZEB1 binds to the DDX56 promoter and facilitates transcription of DDX56. (A) HEK293T cells were co-transfected with the DDX56-luciferase construct together with the indicated plasmids for 48 h. (B-C) mRNA (B) and protein (C) levels of indicated genes were detected using RT-PCR and western blotting, respectively. (D) MHCC97H and Huh-7 cells were co-transfected with DDX56 promoter luciferase constructs and ZEB1-silencing molecules. (E) Computational prediction of ZEB1 binding sites on DDX56 promoter region (-2000 to +1) by JASPAR and MEM-suite matrix model. (F) ChIP-PCR assay was performed using anti-ZEB1 to confirm the two predicted ZEB1-binding sites on the DDX56 promoter. (G) EdU assay showed that ectopic DDX56 expression in ZEB1-silenced cells partially reversed the ZEB1 loss-of-function-mediated inhibitory effect on HCC proliferation. Scale bars: 100 µm. (H-I) Protein levels of ZEB1 and DDX56 in HCC tissues and corresponding adjacent normal tissues (n = 24) were examined using western blot analysis (H), followed by an analysis of their correlation (r = 0.4435, P = 0.03) (I). Protein band intensity was quantitated using Image J software. (J) A schematic model showing the ZEB1-DDX56-MIST1 axis regulating HCC proliferation. ZEB1 directly binds to the promoter region of DDX56 and promotes its transcription. Upregulated DDX56 enhances MIST1 transcription by recruiting MECOM and mono-methylating H3K9 at the MIST1 promoter, which subsequently reduces PTEN protein and facilitates AKT phosphorylation, to promote HCC proliferation. Importantly, the PTEN agonist, Oroxin B (OB), blocked the DDX56-mediated PTEN-AKT signaling pathway, suggesting that treating of HCC patients with OB may result in a beneficial therapeutic intervention.

Fig 5: DDX56 promotes MIST1 transcription by recruiting the epigenetic regulator MECOM. (A) Co-immunoprecipitation experiment showed the interaction between DDX56 and MECOM in MHCC97H and Huh-7 cells. (B) Immunofluorescence analysis shows the co-localization of DDX56 (green) and MECOM (red). (C) DDX56 rescues MECOM to regulate MIST1 promoter-driven luciferase activity. DDX56-knockdown HCC cells were co-transfected with MIST1 promoter luciferase constructs and MECOM overexpression plasmid. Cell lysates were measured for firefly and Renilla (RL-TK) activity. (D-E) ChIP-qPCR assay with anti-MECOM or anti-H3K9me1 was performed in MHCC97H and Huh-7 cells. DDX56 loss-of-function reduced MECOM (D) or H3K9me1 (E) occupancy at the promoter of MIST1 gene (top). Agarose gel electrophoresis of PCR fragments after ChIP (bottom). (F) Western blot analysis of MIST1/PTEN/AKT axis in DDX56-silenced HCC cells, and MECOM overexpression reversed the effect of DDX56 on the MIST1/PTEN/AKT axis. (G) EdU assays revealed that ectopic MECOM expression in DDX56-silenced cells partially reversed the DDX56 knockdown-mediated inhibitory effect on HCC proliferation. Scale bars: 100 µm.