Fig. 2: SNORD88B promotes the self-renewal of liver CSCs.
A Northern blot showed SNORD88B depletion efficiency in HCC cell lines and HCC primary cells. Overexpression of SNORD88B (oe88B) rescued the expression of SNORD88B. shVec, shRNA empty vector; sh88B, depletion of SNORD88B. B Sphere-formation assays of depleted or rescued SNORD88B HCC cells. Statistical results of sphere-formation ratios were calculated by serial sphere-formation assays. Scale bar, 500 μm. n = 3 biologically independent experiments. Exact P values from left to right: 0.001044, 1.12E−05, 0.000877, 5.62E−05, 1.86E−05, 0.000109. Data are shown as means ± SD. C Gradient numbers of SNORD88B depletion and control cells were subcutaneously injected into BALB/c nude mice (8 W) for 3 months to detect tumor initiation. The typical tumor images were displayed in the left panel and the ratios of tumor-free mice were shown in the right panel. n = 7 for each group. Scale bar, 1 cm. Exact P value: 0.0139. D 1 × 106 SNORD88B depleted or control cells were subcutaneously injected into BALB/c nude mice (8W), followed by measurement of tumor progression every 4 days. n = 5 for each group. Results are shown as means ± SD. Exact P values from left to right: 0.00177, 7.88E−05, 0.00037, 1.43E−05. E Orthotopic liver tumor growth was imaged via luciferase signals. Representative images are shown in the left panel, and statistical results are shown as means ± SD (right panel). n = 5 for each group. Exact P values from left to right: 0.0006, 0.0004. F The effect of silencing SNORD88B expression through ASOs was verified in Huh7 cells. n = 3 independent experiments. Data are shown as means ± SD. Exact P values from left to right: 0.00045, 1.74E−05. G, H Primary HCC cells were subcutaneously injected into BALB/c nude mice. 25 mg/kg ASOs were injected around the tumors on days 24, 26, 28, 30 and 32. Mice were sacrificed on the 40th day after injection, and the tumors were excised (G) and weighed (H). Representative images were shown. Data are shown as means ± SD. n = 5 mice per group. Scale bar, 1 cm. Exact P value: 1.41E−05. I In vivo tumorigenic cell frequency assay. Tumor-free mice are counted (right panel) and representative tumor images are shown (left panel). n = 7 for each group. Exact P value: 2.19E−05. J 1 × 106 SNORD88B-overexpressing or control cells were subcutaneously injected into BALB/c nude mice, followed by measurement of tumor progression every 4 days. n = 5 for each group. Results are shown as means ± SD. Exact P values from left to right: 0.0163, 0.0011, 0.00016, 0.0021. K Orthotopic liver tumor growth was imaged via luciferase signals. Representative images are shown in the left panel, and statistical results are shown as means ± SD (right panel). Immunodeficient NSG mice were used. n = 5 for each group. Exact P value: 0.000278. *P < 0.05; **P < 0.01; ***P < 0.001 by two-tailed Student’s T-test. Data are representative of at least three independent experiments.
