Fig. 4: miR-22 binds the 3′UTR of MYC to inhibit its expression; MSI2 antagonizes MIR22HG processing.

A Hypothetical model for MSI2 regulation of MYC via inhibition of mature miR-22. B Anti-miR-22 restored MYC expression. PH5CH cells were transiently transfected with either of two different anti-miR-22 oligonucleotides or combined anti-miR-22s as indicated. Cells were lysed 72 h after transfection and immunoblots were probed with MYC antibodies. C MYC levels were reduced by miR-22 but MSI2 reversed miR-22 suppression of MYC. Huh7 cells were transiently transfected with miR-22 mimic oligonucleotides, or with appropriate control miR oligonucleotides as indicated. Cells were lysed 72 h after transfection and immunoblots were probed with MYC antibodies. D PH5CH cells were transfected with increasing amounts of synthetic MSI2 expression vector or a vector control. Total RNA was isolated as described in methods for northern blotting and probed with miR-22 and 5 S RNA probes as indicated. A separate aliquot of cells was lysed and analyzed by western blotting with specific antibodies as indicated. E Mouse TICs were transfected with scramble shRNA or shRNA targeting MSI2. Cells were processed and examined by northern and western blotting as above. Northern blot revealed the reduction of mature miR-22 in HCC cells. F miR-22 repressed MYC 3′UTR-driven luciferase activity. Mutation of putative Target 2 miR-22 binding site abrogated repressive effect of miR-22. Activities were expressed as relative luminescence units (RLU) normalized to the activity of co-transfected Renilla luciferase. Data represent mean ± S.D. from three independent experiments. *P < 0.05. G A hypothetical model shows that MSI2 binding to MIR22HG and MYC mRNA suppresses miR-22 maturation and stabilizes mRNA, rendering unimpeded translation of MYC mRNA to promote self-renewal of TICs and liver oncogenesis. Overexpression of MSI2 inhibits MIR22HG RNA processing. H RT-qPCR analysis of mature miR-7014-3P and Msi2 mRNA expression in Hepa1-6 murine HCC cell line stably transduced with sh-scramble or sh-Msi2 (Left panel). Protein levels for Vegfa, Myc, Jun, Msi2 in Hepa1-6 ectopic expressing Msi2 or stably transduced with sh-Msi2 was analyzed by immunoblotting (Center and Right panels). Data represent mean ± S.D. from three independent experiments. *P < 0.05. I Protein level for Vegfa, and Msi2 in Hepa1-6 ectopic expressing either vector or miR-7014-3P-sponge (Left panel) or miR-ctrl or miR-7014-3P mimic (Right panel) in the presence or absence of MSI2 expression was analyzed by immunoblotting. J Schematic representation of Vegfa 3′-UTR luciferase reporter plasmids wild-type (WT) and mutant (MUT) with wild-type or mutant of miR-7014-3P binding sites, as indicated respectively (Left panel). Hepa1-6 cells stably transduced with sh-scramble or sh-Msi2 were co-transfected with vector or miR-7014-3P-sponge, and Vegfa 3′-UTR-WT or -MUT luciferase reporter plasmids, as indicated. Relative luciferase activities were measured 24 h post-transfection (right panel). Data represent mean ± S.D. from three independent experiments. *P < 0.05.