Fig. 7

Epigenetic modulation of TGIF1 and targeting HAS2 suppresses TGIF1 loss-induced PDAC cell migration in vitro. a i, Immunoblot analysis of HDAC1, HDAC2, HDAC3, DNMT1, and HAT1 from PKP and PKTP PDAC cells. ii, Increase of histone H3K27 acetylation induced by TGIF1 loss binding on EMT associated SNAIL1, ZEB1 and COL1A1 promoter DNA of PKTP cells. Values are from three independent experiments with RT qPCR in duplicate. Bars, SD. IgG (2 μg/IP) was used as a negative IP control. The treatment with the commercial HDAC inhibitor SAHA (10 μM; 2 days) served as positive control for anti-acetyl-Histone H3K27 (Lys27) CHIP analysis. *P < 0.01. b Detection of TFF1, CDH1, CLDN3, PPARγand P16 promoter methylation statuses by using MSP in PKP and PKTP PDAC cells. c Determination of TGIF1 protein (i) and mRNA (ii) levels in different human PDAC cells through Western blot and RT-PCR analyses. d MSP analysis of the promoter region of TGIF1 in indicated human PDAC cell lines. UM, unmethylated primer pair; M, methylated primer pair. e The represented images of methylation status of the TGIF1 promoter in two pairs of human PDAC (tumor) and its adjacent normal pancreatic tissues (normal) by using MSP analysis (i), and their corresponding gene expression levels were determined by RT-qPCR(ii). The housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as an internal control. f Effects of DNA methylation and histone acetylation on the regulation of TGIF1 gene expression. Effects of 5-Aza and SAHA on TGIF1 expression in PDAC cells. Restoration of TGIF1 expression in mouse 6784, 2778, human BxPC-3 and AsPC-1 cells treated with 5-Aza, SAHA alone or both were analyzed using western blot analysis, and β-actin used as loading controls. g western blot analysis of CD44, total and phosphorylated Stat3 in PKTP PDAC cells with or without 4-Methylumbelliferone (4-MU) treatment. h Representative flow cytometric analysis of cell cycle showing PKP and PKTP PDAC cells following 4-MU (10 μM) overnight treatment compared with methanol control groups. i Quantitative assessment of wound closure rates of PKTP PDAC cells were analyzed using wound healing assays with the treatment of 5 μM of the Has2 inhibitor, 4-MU. j Quantitative determination of transwell migration assays of PKP or PKTP PDAC cells treated or untreated with 4-MU (5 μM) for 18 h **p < 0.01; ***p < 0.001. k Effect of 4-MU on PDAC formation in vivo. i. Representative anatomical images of the abdomen of PKP and PKTP mice treated intraperitoneally with 4-MU or vehicle (DMSO) for 4 weeks showing the reduction of PDAC formation in the 4-MU treated PKP and PKTP mice, but not in the mice receiving DMSO treatment. Macroscopic appearance and H&E histological analysis of murine pancreas and liver after 4-MU treatment or DMSO control groups are shown. Scale bar, 50 μm. ii, Kaplan-Meier survival curves of PKP or PKTP mice with or without 4-MU. PDAC mice treated with 4-MU significantly live longer than untreated mice. *P < 0.05; **P < 0.01 l The schematic diagram showing the roles of TGIF1 in the TGFβ1/Smad signaling in modulating EMT program and tumor metastasis