Fig 2: miR-497 Directly Targets SALL4(A) Putative binding sites of miR-497 in the SALL4 3' UTR of different species. (B) SALL4 3' UTR firefly luciferase activity in HEK293T cells. Upper panel: the sequences of wild-type and mutant 3' UTR show the segments cloned into the CMV-promoter-driven luciferase reporter plasmid. Lower panel: pMIR-REPORT and ß-gal gene expression vector, together with miR-497 mimic, anti-miR497, or control vector as indicated, were transfected into 293T cells. Reporter gene activity was determined and normalized in relation to the co-transfected ß-gal activity. The bars represent the average ± mean from three independent experiments. (C) miR-497 and SALL4 mRNA expression in miR-497-overexpressing Hep3B cells and miR-497-knockdown HepG2 cells was analyzed by real-time RT-PCR assay. (D) Protein expression of SALL4 by western blotting analysis in transfected Hep3B and HepG2 cells. (E) Expression levels of miR-497 and SALL4 were negatively correlated in 50 HCC samples, as measured by real-time PCR. The relative expression of miR-497 and SALL4 values (normalized to 18S rRNA) was subjected to Pearson correlation analysis. (F) Detection of the expression of miR-497 and SALL4 in different HCC cell lines by real-time RT-PCR and western blot, respectively; scatterplot indicates the negative correlation between miR-497 and SALL4 expression. Pearson’s coefficient test was performed to assess statistical significance. *p < 0.05; **p < 0.01; ***p < 0.001.

Fig 3: IL-17B/IL-17RB signaling induces gastric cancer (GC) stemness.Real-time RT-PCR (a) and Western blots (b) analyses of IL-17RB in MGC-803 cells treated with corresponding concentrations of rIL-17B for 48 h. Real-time RT-PCR analyses of LGR5 (c), Oct4 (d), Sall4 (e), and Sox2 (f) mRNA levels in MGC-803 cells treated with corresponding concentrations of rIL-17B for 48 h. (g) Western blots detecting the protein expression of IL-17RB, E-cadherin, N-cadherin, Nanog, Sox2, and Oct4 in IL-17RB knockdown cells and control MGC-803 cells treated with corresponding concentrations of rIL-17B for 48 h, the blots were cropped from different gels but all the gels had been run under the same experimental conditions. (h) MGC-803 cells treated with corresponding concentrations of rIL-17B for 48 h, then induced with adipogenic differentiation medium. Representative images show accumulation of lipid droplets after undergoing adipogenic differentiation.

Fig 4: miR-497 Regulates HCC Metastasis and Self-Renewal(A) Hep3B and HepG2 cells transduced with miR-497 mimic, anti-miR497 vector, or control vector as indicated were subjected to cell migration and invasion assays. Migrated/invaded cells in fields were quantified, and representative photographs are shown. Data are represented as mean ± SEM. *p < 0.05; **p < 0.01. (B) Wound healing assay was performed to determine the cell migratory capacity. (C) Tumor sphere-forming capacity in HCC cell lines as indicated was analyzed by tumor sphere formation assay; the right panel indicates statistical results as means ± SEM. (D) qRT-PCR analysis of the mRNA expression of SALL4, CDH1, vimentin, Nanog, Oct4, and Bmi-1 in HepG2/Ctrl and HepG2/Anti-miR-497 cells. (E) Western blot analysis of SALL4, E-cadherin, vimentin, Oct4, Bmi-1, and Nanog expression in Hep3B/Ctrl and Hep3B/miR-497 cells, as well as in HepG2/Ctrl and HepG2/anti-miR-497 cells. (F) SALL4 knockdown abolished sphere formation ability reduced by miR-497. Representative sphere formation is indicated in the left panel. Relative tumor sphere numbers were calculated as means ± SEM (right panel). (G and H) SALL4 knockdown abolished migration and invasion capacity down-regulated by miR-497 as analyzed by Transwell assay (G) and wound healing assay (H). (I) The expression of E-cadherin, vimentin, Oct4, Bmi-1, and Nanog was detected in SALL4 KD/Ctrl and SALL4 KD/miR497 HCC cells. *p < 0.05; **p < 0.01. Scale bars, 100 µm.

Fig 5: IL-17RB correlates with stemness in gastric cancer (GC) tissues.(a) The correlation of IL-17RB and Oct4 mRNA expression (r = 0.6683, p < 0.0001), (b) IL-17RB and Nanog (r = 0.6292, p = 0.0001), (c) IL-17RB and Lgr5 (r = 0.4914, p = 0.0015), and (d) IL-17RB and Sall4 (r = 0.5396, p = 0.0005). (e) Western blot analyses of IL-17RB, Oct4, Sox2, and Sall4 levels in gastric cancer tissues, the blots were cropped from different gels but all the gels had been run under the same experimental conditions.