Figure 6.

Autocrine/paracrine IL-6 or LIF/gp130/STAT3 pathways regulate H19 expression. (A) Schematic representation of the proximal region of the human H19 gene and RNA (human genome: hg19 ch12). Histogram images of H3K27me3 and H3K27Ac in PANC-1 cells were obtained from chromatin immunoprecipitation (ChIP)-atlas (http://chip-atlas.org/, accessed on 18 January 2022). The red line displays the ICR of H19. Three sets of PCR primers designated as Primer 1 (proximal region), Primer 2 (middle region), and Primer 3 (distal region) on the predicted H19 promoter region were used for analysis of ChIP. (B) ChIP assays for PANC-1 after sphere formation were performed using anti-H3K27me3, H3K27Ac, and p-STAT3 antibodies and control IgG, and this was followed by real-time qPCR using the designed primer sets specific for the H19 promoter. The data were normalized according to the percent input method. Data are presented as means ± SD from three independent experiments (* p < 0.05, ** p < 0.01, *** p < 0.005 vs. adherent cells). (C) Real-time qPCR analysis of H19 in PANC-1 cells cultured for 7 days in 2D (adherent) or 3D (sphere) conditions with or without 1 μM SC144 for 7 days. Results are normalized to values obtained for adherent cells (value = 1). Results are presented as means ± SD from three independent experiments. ** p < 0.01 (D) Real-time qPCR analysis of H19 in PANC-1 cells cultured for 7 days in 3D (sphere) conditions after siRNA transfection. Results are normalized to values obtained for siCont transfected sphere cells (value = 1). Results are presented as means ± SD from three independent experiments (**p < 0.01 vs. siCont transfected sphere cells). (E) Schema of epigenetic regulation on the predicted H19 promoter region during sphere formation of PDAC cells. In the adherent state, the H19 promoter region is closed and the H19 expression level is low, while in the sphere formation state, this promoter is activated and p-STAT3 can access this region to facilitate the induction of H19 expression.