Fig 2: Kaplan-Meier curves of overall survival and progression-free survival of different quartiles of WIF1 methylation level in CS. (A) Kaplan-Meier curves of OS showed a significant difference across the Q1, Q2, Q3, and Q4 groups (p = 0.016). (B) Kaplan-Meier curves of PFS showed a weak difference among these four groups, but was not significant (p = 0.094). (C) OS of Q1 group was significantly shorter than the Q2–4 group (p = 0.008). (D) PFS of Q1 group was significantly shorter than the Q2–4 group (p = 0.023). Q1 represents the “high-methylation-level group” and Q2, Q3, and Q4 collectively represent the “low-methylation-level group”.

Fig 3: SLC16A1-AS1 increases WIF1 expression via sponging miR-552-5p. (A) Illustration of the prospective binding sites of miR-552-5p in WIF1 3′-UTR predicted with miRanda (http://www.microrna.org/). (B) mRNA expression of WIF1 was significantly lower in BC cells (MDA-MB-231 and MCF7) than in normal mammary gland epithelial cells (MCF 10A) analyzed by qRT-PCR. (C) Dual-luciferase activities in MDA-MB-231 and MCF7 cells with negative control (NC), wild type (WT), or mutated (mut) reporter of WIF1 3′-UTR with miR-552-5p or without (NC) miR-552-5p mimics. (D) mRNA and protein expressions of WIF1 in MCF7 and MDA-MB-231 cells after miR-552-5p was silenced (miR-552-5p inhibitor) or overexpressed (miR-552-5p mimics). (E) mRNA (left panel) and protein expressions (middle and right panels) of WIF1 in SLC16A1-AS1 overexpressed in MDA-MB-231 and MCF7 cells with or without co-transfecting miR-552-5p. (F) Spearman correlation assay of the correlation between WIF1 and SLC16A1-AS1 or miR-552-5p in BC tissues from 80 BC patients (the same specimens used in Figure 1A ). * p < 0.05, ** p < 0.01, *** p < 0.001.

Fig 4: Indirubin inhibits the expression of DNMT1, restores wif-1 expression, and inhibits wnt/β-catenin signal pathway. (a): The expression of wif-1 was promoted and the expression of DNMT1, Frizzled2, Frizzled5, and phosphorylation β-catenin was suppressed after treatment with different concentrations (0.04 μM, 0.2 μM, and 1 μM) of indirubin for 48 h compared with the negative control group in HaCaT cells by Western blotting using GAPDH as an internal control. (b): The mRNA expression levels of related proteins after treated with different concentrations (0.04 μM, 0.2 μM, and 1 μM) of indirubin. The mRNA expression level of wif-1 was promoted, and the mRNA expression levels of DNMT1, Frizzled2, Frizzled5, and β-catenin were suppressed compared with the negative control group in HaCaT cells by qRT-PCR. (c): WIF-1 promoter methylation level decreased after treated with low (0.04 μM), medial (0.2 μM), and high(1 μM) concentrations of indirubin while abnormal methylation was observed in the negative control group. (d): The silencing of DNMT1 suppresses wif-1 promoter hypermethylation in HaCaT cells, likewise, wif-1 promoter hypermethylation was suppressed after treated with indirubin(1 μM) together with si-NC, further, wif-1 promoter hypermethylation was significantly suppressed after treated with indirubin(1 μM) together with si-DNMT1, relative to the si-NC group (Fig.1e). (e): The overexpression of DNMT1 significantly promoted wif-1 promoter methylation level in HaCaT cells, nevertheless,wif-1 promoter methylation level was suppressed after treated with indirubin(1 μM) together with control,wif-1 promoter methylation level was promoted after treated with indirubin(1 μM) together with DNMT1, relative to the control group. (f): The protein expression of wif-1 was encouraged after treated with different concentrations (0.04 μM, 0.2 μM, and 1 μM) indirubin compared with the negative control group in HaCaT cells by ELISA. (g): The mRNA expression of DNMT1 was suppressed after treated with different concentrations (0.04 μM, 0.2 μM, and 1 μM) of indirubin compared with the negative control group in HaCaT cells by ELISA. (d): A: si-NC, B: si-DNMT1, C:si-NC + indirubin, D:si-DNMT1 + indirubin. (e): A: Ctrl, B: DNMT1, C: Ctrl+indirubin, D: DNMT1+ indirubin. *P < 0.05, **P < 0.01, ***P < 0.001. U: unmethylated; M: methylated (N = 3)

Fig 5: The effect of indirubin on transcriptional activity in wif-1 and β-catenin. (a) Luciferase reporter system was used to detect wif-1activity, the results showed concentration raise of pRL-TK-WIF-1-Luc dual luciferase activity after treatment by indirubin(1 μM) compared to the activity in the negative control group, inversely, the concentration reduces of pRL-TK-DNMT-1-Luc dual luciferase activity after exogenously added DNMT1 in HaCaT cells compared to the activity in the negative control group. (b) TOP Flash and FOP Flash reporters are widely used to evaluate the β-catenin-dependent wnt/β-catenin signal. Wnt/β-catenin signal activity was inhibited when treatment with indirubin(1 μM), similar results was observed when exogenously added wif-1. **P < 0.01, ***P < 0.001. (N = 3)