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Fig. 6 | Cancer Cell International

Fig. 6

From: LncRNA CTD-3252C9.4 modulates pancreatic cancer cell survival and apoptosis through regulating IFI6 transcription

Fig. 6

LncRNA CTD-3252C9.4 inhibits IFI6 expression through binding with its transcription factor IRF1. A Cytoplasm and nuclear distribution of CTD-3252C9.4 in pancreatic cancer cells detected by fractionation of Panc-1 and MIApaca-2 cells followed by qRT-PCR. U6 and GAPDH RNAs served as the positive control for nuclear gene expression and cytoplasmic gene expression, respectively. B Luciferase activity was measured in Panc-1 cells co-transfected with pcDNA-Ctrl, pcDNA-CTD-3252C9.4, si-Ctrl, or si-CTD-3252C9.4 and PGL3-IFI6 vectors which included IFI6 promoter. C Left: 11 potential transcription factors of IFI6 were predicted by both JASPAR and PROMO database. Right: Luciferase activity was measured in Panc-1 cells co-transfected with PGL3-IFI6 vectors and vectors expressing the above 11 genes. D Luciferase activity was measured in Panc-1 cells co-transfected with pcDNA-Ctrl, pcDNA-IRF1, si-Ctrl, or si- IRF1 and PGL3-IFI6 vectors. Relative IFI6 mRNA (E) and protein (F) levels in IRF1-overexpressing or IRF1-knocked down Panc-1 cells. G Spearman correlation analysis of the fold change of IFI6 mRNA and IRF1 mRNA in 1104 human breast cancer tissues in starBase public database from TCGA project. H Putative IRF1 binding sites on the promoter region of IFI6. I Mutagenesis in the putative binding site of − 72 ~  − 61 bp fragment of the IFI6 promoter abrogated the induction activity of RUNX2 in the Panc-1 cells. J Luciferase activity was measured in Panc-1 cells co-transfected with pcDNA-Ctrl plus si-Ctrl, pcDNA-IRF1 plus si-Ctrl, pcDNA-IRF1 plus pcDNA-CTD-3252C9.4, or pcDNA-IRF1 plus si-CTD-3252C9.4 and PGL3-IFI6 vectors. Relative IFI6 mRNA (K) and protein (L) levels in Panc-1 cells transfected with pcDNA-Ctrl plus si-Ctrl, pcDNA-IRF1 plus si-Ctrl, pcDNA-IRF1 plus pcDNA-CTD-3252C9.4, or pcDNA-IRF1 plus si-CTD-3252C9.4. M The possibility of interaction between CTD-3252C9.4 and IRF1 was predicted by RPISeq predictions. N CatRAPID fragments module prediction of the interaction profile and matrix between CTD-3252C9.4 and IRF1. O The interaction between CTD-3252C9.4 and IRF1 were verified by RNA pulldown assays. P The enrichment of IRF1 on the IFI6 promoter relative to IgG in control, CTD-3252C9.4-overexpressing or CTD-3252C9.4- knocked down Panc-1 cells detected by ChIP-qPCR assays. Q A working model for the role of IRF1-targeted CTD-3252C9.4 in pancreatic cancer proliferation and apoptosis. During pancreatic tumorigenesis, silencing of CTD-3252C9.4 facilitated proliferation and inhibited apoptosis of pancreatic cancer cells through up-regulating the expression of IFI6 via binding with its transcription factor IRF1. All data are shown as the mean ± SEM. *P < 0.05 and **P < 0.01 by two-tailed Student’s t-test

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