Fig. 5: miR-183-5p.1 inhibits liver T-ICs expansion via targeting MUC15.

A A potential target site for miR-183-5p.1 in the 3′UTR of human MUC15 mRNA, as predicted by the program TargetScan. To disrupt the interaction between miR-183-5p.1 and MUC15 mRNA, the target site was mutated. B Luciferase reporter assays performed in miR-183-5p.1 overexpression and control HCC spheres transfected with wild-type or mutant MUC15 3′UTR constructs. C Real-time PCR analysis of MUC15 in spheroids generated from miR-183-5p.1 overexpression hepatoma cells and control cells. D Western blot analysis of indicated proteins in spheroids generated from miR-183-5p.1 overexpression hepatoma cells and control cells. E The correlation between the level of MUC15 and miR-183-5p.1 in primary HCC cells (n = 30) was determined by real-time PCR analysis. F Representative images of hepatoma spheroids generated from miR-183-5p.1 overexpression hepatoma cells and control cells. The number of spheroids was counted and compared. G Hepatoma cells dissociated from Hep3B miR-183-5p.1 overexpression spheroids or control spheroids were inoculated into NOD-SCID mice subcutaneously, and the tumorigenicity was evaluated two months post inoculation. H Hep3B/Huh7 miR-183-5p.1 and control cells infected with MUC15 overexpression virus were subjected to western blot assays. I Hep3B/Huh7 miR-183-5p.1 and control cells infected with MUC15 overexpression virus were subjected to spheroids formation. J Hep3B/Huh7 miR-183-5p.1 and control cells infected with MUC15 overexpression virus were subjected to in vitro limiting dilution assays. K Hep3B miR-183-5p.1 and control cells infected with MUC15 overexpression virus were subjected to in vivo limiting dilution assays. Tumors were observed over 2 months; n = 8 for each group. All results are presented as the mean ± SD, and statistical significance was assessed using a two-tailed Student t-test. *p < 0.05.