Fig 2: The mRNA expression and methylation analysis of DACT1, DACT2 and DACT3 in esophageal cancer cell lines. a, b, c Schematic structure of CpG islands in DACT1, DACT2 and DACT3 genes. The MSP and BGS-regions were shown in the schematic. The transcription start point was as +1. d The changes of DACT1, 2, 3 mRNA expression in treated or untreated cells with 5-Aza-Dc or TSA by RT-PCR method; e, f, g Relative mRNA expression of DACT1, 2, 3 in treated or untreated cells with 5-Aza-Dc or TSA by Quantitative realtime RT-PCR method. h The methylation status of DACT1, 2, 3 in four cell lines with(+) or without(−) 5-Aza-Dc treatment detected by MSP method. M, methylated gene; U, unmethylated gene. i The methylation status of every CpG site in two regions of DACT1, one region of DACT2 and one region of DACT3 in four tumor cell lines by BGS method. The color of circles for each CpG site represents the percentage of methylation. The “F” and “R” represent forward and reverse primers for MSP, respectively

Fig 3: miR-181a inhibits DACT2 by downregulating methylase MLL3 to activate YAP-VEGF pathway in HUVECs(A) Intersection of Venn diagram to show target genes of miR-181a-5p predicted by the online databases Targetscan, miDIP, RAID, StarBase, miRWalk, and miRDB. (B) The binding sites between miR-181a and MLL3 predicted via TargetScan. (C) Binding relationship between miR-181a and MLL3 determined by dual-luciferase reporter gene assay. (D) mRNA expression of MLL3 in HUVECs after corresponding miR-181a treatment analyzed by quantitative real-time PCR. (E) Protein expression of MLL3 in HUVECs after corresponding miR-181a treatment analyzed by western blot. (F) mRNA expression of MLL3 and DACT2 in HUVECs after alteration of MLL3 determined by quantitative real-time PCR. (G) Protein expression levels of MLL3, H3K4me1, and DACT2 in HUVECs after alteration of MLL3 determined by western blot. (H) MLL3 and H3K4me1 expression in DACT2 enhancer in HUVECs cells after alteration of MLL3 analyzed by ChIP assay. (I) mRNA expression of DACT2 in HUVECs after corresponding miR-181a treatment determined by quantitative real-time PCR. (J) Protein expression of DACT2 in HUVECs after corresponding miR-181a treatment analyzed by western blot. (K) Protein expression of H3K4me1, YAP, phosphorylated YAP, and VEGF in HUVECs after corresponding miR-181a treatment determined by western blot. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 versus HUVECs transfected with mimic-NC or si-NC; #p < 0.05, ##p < 0.01, ###p < 0.001 versus HUVECs transfected with inhibitor-NC or vector-NC. Data were presented as mean ± SD. Unpaired t test was used to compare two sets of data. One-way ANOVA with Tukey post hoc test was used to analyze data from multiple groups. The cell experiments were repeated in triplicate.

Fig 4: Exosomal miR-181a from hypoxia-treated PTC cells stimulates proliferation, migration, and angiogenesis in HUVECs(A) HUVEC proliferation in the presence of PBS, normoxic-EXs, or hypoxia-EXs determined by EdU assay (×200). (B) HUVEC migration in the presence of PBS, normoxic-EXs, or hypoxia-EXs analyzed by transwell assay (×200). (C) The formation of network-like structures by HUVECs in the presence of PBS, normoxic-EXs, or hypoxia-EXs (×200). (D) VEGF concentration in culture medium in the presence of PBS, normoxic-EXs, or hypoxia-EXs determined by ELISA. (E) mRNA expression of MLL3 and DACT2 in HUVECs in the presence of PBS, normoxic-EXs, or hypoxia-EXs analyzed by quantitative real-time PCR. (F) Protein expression of MLL3, H3K4me1, DACT2, YAP, phosphorylated YAP, and VEGF of HUVECs in the presence of PBS, normoxic-EXs, or hypoxia-EXs analyzed by western blot. (G) miR-181a expression in BCPAP cells treated with miR-181a inhibitor detected by quantitative real-time PCR. (H) miR-181a expression in hypoxia-EXs/miR-181a inhibitor detected by quantitative real-time PCR. (I) HUVEC proliferation in the presence of hypoxia-EXs/miR-181a inhibitor tested by EdU assay (×200). (J) HUVEC migration in the presence of hypoxia-EXs/miR-181a inhibitor analyzed by transwell assay (×200). (K) The formation of network-like structures by HUVEC in the presence of hypoxia-EXs/miR-181a inhibitor (×200). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 versus HUVEC treated with PBS or hypoxia-EXs/inhibitor-NC. Data were presented as mean ± SD. Unpaired t test was used to compare two sets of data. One-way ANOVA with Tukey post hoc test was used to compare data from multiple groups.

Fig 5: Kaplan–Meier univariate survival analysis of DACT1 / DACT2 expression and methylation status in ESCC. a, b, c Kaplan–Meier curves for cumulative survival stratified by DACT1 and DACT2 expression: showing a direct correlation between DACT1 or DACT2 expression and ESCC patient’s survival. d, e, f, g Kaplan–Meier curves for cumulative survival stratified by the methylation status of two regions of DACT1 and one region of DACT2: There was not correlation between the region 1 methylation status of DACT1 and the ESCC patient’s survival. The hypermethylation of TSS-region in DACT1 and DACT2 were associated with the poor patient survival of ESCC cases; Simultaneous methylation of DACT1 and DACT2 showed worst prognosis. (H) Kaplan–Meier curves for cumulative survival stratified by methylation status and UGIC family history: ESCC patients with positive UGIC family history and DACT1 or DACT2 methylation showed poor patient survival. (I) Kaplan–Meier curves for cumulative survival stratified by methylation status and TNM stages: Stage III and IV ESCC patients with DACT1 or DACT2 methylation showed the poor patient survival