Fig 2: EZH2 recruitment and epigenetic modifications onto promoter of cell-adhesion molecules upon combined treatment or H19 silencing. (a–c) Recruitment of EZH2 and H3K27me3 level on the promoter region of CDH1 (a), ITGB3 (b), and ITGB4 (c) by ChIPs after 18 h treatment with estrogen (E2, 10-7 M), 1% O2 hypoxia, alone or in combination in PCa cells (C27IM). No antibody (NoAb) served as negative control. Values represent mean ± SEM of three independent experiments. * p < 0.05 vs. NT, $ p < 0.05 vs. E2, # p < 0.05 vs. Hyp. (d) In vivo H19 interaction with polycomb subunit EZH2 before and after Hyp+E2 (18 h) detected by RNA-ChIP assays. IgG was used as control. Immunoprecipitated RNA was recovered and analyzed by qRT-PCR. The results are expressed as mean ± SEM of three independent experiments. * p < 0.05 EZH2 vs. IgG, $ p < 0.05 Hyp+E2 vs. NT. (e) Recruitment of EZH2 and H3K27me3 level on the promoter region of CDH1, ITGB3, ITGB4, and RUNX2 by ChIPs upon H19 silencing (siH19) compared with control (scamble, scr) in PCa cells (C27IM). Non-specific immunoglobulin (IgG) served as negative control. Values represent mean ± SEM of three independent experiments. * p < 0.05 vs. scramble.

Fig 3: H19 mediates transcriptional repression of cell-adhesion molecules. (a–c) H19 (a); E-cadherin (CDH1) (b); ITGB3, ITGB4, ITGA2, and RUNX2 (c) mRNA levels quantified by qPCR in C27IM transfected with siRNA specific to H19 (siRNA H19) or scramble. Data, plotted as fold induction siH19 vs. scramble, represent mean ± SEM of three experiments. * p < 0.05 vs. scramble. (d) Protein analysis of E-cadherin, ß3, and ß4 integrin before and after siRNA H19 by Western blot. Tubulin or ß Actin served as control. Molecular weight marker is indicated. Upper panels: representative experiments. Lower panel: densitometric analysis, reported as fold induction vs. scramble, represent mean ± SEM of three experiments. * p < 0.05 vs. scramble. (e) CDH1, ITGB3, ITGB4, and RUNX2 mRNA levels quantified by qPCR in C27IM transfected with siRNA specific to H19 (siH19) or scramble for 48 h and treated for additional 18 h with E2 (10-7 M), 1% O2 (HYP), alone or in combination. Data, plotted as fold induction vs. scramble NT, represent mean ± SEM of three experiments. * p < 0.05 vs. scramble NT, ^ p < 0.05 vs. scramble.

Fig 4: Cytotoxic activity of lactoferrin in various cell lines under normoxic (21% O2) or hypoxic (1% O2) conditions. (A) Cytotoxic activity evaluated as IC50 values with the MTT assay. Statistical significance is presented as + +: p < 0.01 (vs. TIG-3), **: p < 0.01 (indicated paired samples). (B) Effects of LF treatment on HIFs (HIF-1a and HIF-2a), EMT markers (E-cadherin, N-cadherin, and vimentin), and p53 proteins in KD and HSC2 under normoxic (N) and hypoxic (H) conditions for 24 h evaluated by using immunoblot analyses. Expression level of ß-actin was used as a loading control. A representative result is shown (n = 3). (C) Effects of LF treatment on expression of HIF-1 target genes, CA9, DEC1, and DEC2. (D) EMT marker genes, CDH1, CDH2, and VIM. (E) Apoptosis and cell cycle related genes, BCL2, BAX, and CDKN1A, in KD and HSC2 under normoxic (N) and hypoxic (H) conditions for 24 h evaluated by using quantitative RT-PCR. Relative gene expression level was calculated by using ACTB expression as the denominator for each cell line (n = 3). For all quantitative values, the average and SD are shown. Statistical significance is represented as +: p < 0.05 and ++: p < 0.01 (N vs H); and #: p = 0.05, *: p < 0.05, and **: p < 0.01 (indicated paired samples).

Fig 5: (A–C) Expression of surfactant protein C (SPC), a-smooth muscle actin (a-SMA), E-cadherin (E-cad) and N-cadherin (N-cad) in RLE-6TN cells. The control group (untransfected RLE-6TN cell monolayer), Runx3 group (transfected with Runx3 overexpression plasmid for 72 h), siRunx3 group (Runx3 deficient RLE-6TN cell monolayer transfected with siRNA of Runx3 for 72 h), transforming growth factor-ß1 (TGF-ß1) group (2.5 ng/ml TGF-ß1 was used to treat the RLE-6TN cell monolayer followed by culture for 48 h), Runx3 + TGF-ß1 group (transfected by Runx3 overexpression plasmid for 24 h, then 2.5 ng/ml TGF-ß1 was used and cultured for the next 48 h). Values are presented as the means ± SD; **P<0.01 vs. control group.