Table 1.
The characteristics of patients.
Table 2.
Primer sequences for real-time PCR.
Fig 1.
Gli1 predicts poor prognosis in AML and Shh-Gli1 signaling is activated in MDS.
(A). Kaplan-Meier plots of overall survival (OS) in patients with AML, stratified by Gli1 expression. Data was obtained from the Cancer Genome Atlas (TCGA) database [23] (r = 1.41, P = 0.075). (B). Real-time PCR analysis of DNMT1, Gli1, Smo, and Shh in primary CD34+cells (n = 32) from MDS and acute myeloid leukemia associated with MDS marrow samples. (C). A statistically significant positive correlation of DNMT1 and Gli1 expression in MDS (Spearman’s correlation analysis, R = 0.563, P < 0.01). (D). Immunofluorescent staining of SHH and GLI1 in primary bone marrow-derived CD34+cells from MDS patients and control healthy donors.
Fig 2.
Knockdown of Gli1 induces MUTZ-1 cell apoptosis and cell cycle arrest.
Gli1 knockdown MUTZ-1 cells were cultured in the presence (500 ng/ml) or absence of Shh-N for 48h. (A). Representative images of cell cycle distribution (left panel), and the percentage of cells in the G0/G1, S, or G2/M phases of cell cycle is indicated in the bar charts (right panel). (B). Representative images indicating cellular apoptosis determined by flow cytometry (left panel), and the sum percentage of early and late apoptotic cells indicated in the bar charts (right panel). All experiments were performed in triplicate and the results are expressed as average ± SEM. *P < 0.05.
Fig 3.
Knockdown of Gli1 inhibits in vitro and in vivo MDS cell growth.
(A). CCK8 assays were performed to determine the proliferation of MUTZ-1 cells transfected with sh-Gli1 (Gli1 sh1 and Gli1 sh2) and control lentiviral vector (Scramble), wherein the knockdown of Gli1 was found to suppress MUTZ-1 growth in vitro. Two-way ANOVA with post hoc analysis (Bonferroni test) was performed to determine the effects of Gli1 knockdown and multiple time points on cell viability. (B). Representative images of tumor growth in nude mice. (C). Effects of Gli1 knockdown on tumor growth in vivo, wherein tumors developed from MUTZ-1 cells stably transfected with shGli1 showed smaller tumor volume and weights. *P < 0.05. Results are expressed as mean ± SEM.
Fig 4.
Gli1 silencing enhanced the demethylating effect of 5-aza-dC on p15 and subsequently promoted p15 expression.
(A). Relative expression of DNMT1 and p15 mRNA upon Gli1 knockdown was analyzed by real-time PCR. Expression levels of beta-actin (ACTB), used as a housekeeping gene, were taken as control. Fold-change was calculated with the 2−ΔΔCt method compared with controls. All experiments were performed in triplicate and the results are expressed as average ± SEM. *P < 0.05. (B). Western blot analysis of the effects of Gli1 knockdown on DNMT1 and p15 protein expression. Each sample was normalized to the respective related beta-actin (ACTB) expression. (C). Methylation-specific PCR for identification of changes in the methylation status of p15 promoter in MUTZ-1 cells transfected with sh-Gli1 (Gli1 sh1 and Gli1 sh2) and control lentiviral vector (Scramble) treated with or without 5-aza-dC (2 μM) for 48h. Images represent PCR-amplified products separated on 2% agarose gels and visualized under UV light after staining with ethidium bromide.
Fig 5.
Gli1 silencing significantly sensitizes 5-aza-dC to inhibit MUTZ-1 cells.
Gli1 silenced and scramble MUTZ-1 cells were cultured with or without 5-aza-dC (2 μM) for 48 h. (A). CCK8 assays were performed to determine the proliferation of MUTZ-1 cells transfected with sh-Gli1 (Gli1 sh1 and Gli1 sh2) and control lentiviral vector (Scramble) in the presence of different concentrations of 5-aza-dC. Two-way ANOVA with post hoc analysis (Bonferroni test) was performed to determine the effects of Gli1 knockdown and 5-aza-dC on cell viability. (B). Representative images of cell cycle distribution (left panel), and the percentage of cells in the G0/G1, S, or G2/M phases of the cell cycle are indicated in the bar charts (right panel). (C). Representative images of cellular apoptosis determined by flow cytometry analysis (left panel), and the sum percentage of early and late apoptotic cells indicated in the bar charts (right panel). *P < 0.05. Results are expressed as mean ± SEM.