Fig. 5

SOX2 modulates the expression of TUSC3 through miR-181a-5p and miR-30e-5p. a The sequence of the potential binding sites for miR-181a-5p and miR-30e-5p in the 3’-UTR of TUSC3. The sites are conserved in human and rodents. b The expression of TUSC3 is repressed by miR-181a-5p and miR-30e-5p mimics as measured by Western blot analysis. c The application of specific inhibitors against miR-181a-5p and miR-30e-5p increases the expression of TUSC3 in ZR7530 cells. d Schematic diagram of the binding sites for miR-181a-5p and miR-30e-5p in 3’-UTR of TUSC transcript. Mutated binding sites (star) were linked with Luciferase reporter (pMIR-Report-Luc). e Mutations of the potential binding sites for miR-181a-5p and miR-30e-5p lead to increased luciferase activities of TUSC3 reporter. Co-transfection of negative mimics and pMIR-Report-Luc-TUSC3-3UTR-WT construct is used as control (Con). Mic indicates miR-181a-5p or miR-30e-5p mimics. Mics indicates mixture of miR-181a-5p and miR-30e-5p mimics, ab* indicates both of a* and b* potential bindings sites are mutated, cd* indicates both of c* and d* potential bindings sites are mutated, and abcd* indicates all four potential bindings sites are mutated. f SOX2 knockdown leads to increased protein levels of TUSC3. g In xenograft initiated by ZR7530 cells, SOX2 knockdown leads to reduced expression of miR-181a-5p and miR-30e-5p, but not the expression of TUSC3 mRNA. The transcript levels were measured by examining RNA expression in three individual tumour nodules in each group. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001 vs control, and the error bars indicate the mean ± standard deviation (SD)