Fig. 5: MSI2 upregulation and MYC coexpression promote self-renewal, tumor-initiation abilities of TICs and liver oncogenesis.

A MSI2 overexpression increased colony formation whereas shRNA silencing of endogenous MSI2 reduced colony formation in Huh7 cells. Combined MSI2 overexpression with MYC knockdown reduced colony formation compared to MSI2 overexpression only. Quantitation of colony numbers for each treatment (Right panel). Data represent mean ± S.D. from three independent experiments. *P < 0.05. B In vitro self-renewal was tested by spheroid formation assay; overexpressing MSI2 resulted in larger numbers of spheroids as compared to MSI2-silenced TICs. Spheroid formation data are summarized in the histogram (Right panel). Data represent mean ± S.D. from three independent experiments. *P < 0.05. C Flow cytometric analysis for MSI2, MYC and NANOG expression in HepG2 spheroids vs. normal culture cells. Representative dot plots for population analysis of the MSI2⁺, MYC⁺, and NANOG⁺ stemness-enriched cells from three independent experiments. NANOG^high/MSI2^high and NANOG^high/MYC^high- pertinent quadrants are labeled in red squares. D Mice implanted with Huh7 cells transduced with lentiviral vectors expressing either shRNA for MSI2 knockdown or cDNA for MSI2 overexpression. Representative tumor growth at day 42 post-xenograft implantation is shown. E Tumor weights for xenotransplants as per panel (D). Total tumor weights for each animal were recorded weekly as indicated. (mean ± S.D., n = 10) *p < 0.05. F Tumor volume for xenotransplants as per panel (D). Tumor volumes were monitored for up to ten weeks. Surviving mice were euthanized on day 70. (mean ± S.D., n = 10) *p < 0.05. G Xenotransplant tumor histology. H&E staining of representative tumor xenograft tissues is shown. H Summary—Kaplan–Meier plot of remaining mice with tumor volumes <1500 mm³ as indicated. n=number of animals. *p < 0.05. I MSI2 and MYC co-immunostaining of xenograft tumor specimens. Immunofluorescent microscopy demonstrated that increased expression of MSI2 positively correlated with MYC expression in xenograft tissue samples. J Scoring of cells co-staining positively for MSI2 and MYC at 20⨯ magnification, N = 30 microscopic fields for all groups, *p < 0.05. K Immunofluorescent microscopy detection of increased expression and co-localization of MSI2 and MYC in human HCC samples. L Immunoreactivity Score (IRS) of MSI2 in non-tumor and tumor regions of clinical HCC specimens. (Top Row) Percent of staining, the intensity of staining, and Immunoreactivity Score (product of the two) for MSI2 protein in Stages I-II (Top Row) and Stages III-IV (Bottom Row). M Representative immunofluorescent microscopy detection of MYC, MSI2, and TIC marker NANOG in normal and HCC tissues, indicating that NANOG+ TICs have elevated levels of MSI2 and MYC. N Immunoreactivity score (IRS) of MYC, MSI2 and NANOG in normal and HCC tumor tissue, *p < 0.05.