Fig. 8: RUNX1-IT1 regulates C-FOS expression by recruiting RUNX1 to the C-FOS promoter.

a WT and mutant C-FOS promoters were constructed using the pGL3 vector. b, c Luciferase activity assays were performed using PC cells (PANC-1 and SW1990) with RUNX1 or RUNX1-IT1 knockdown and overexpression that were cotransfected with a C-FOS WT promoter. d, e RUNX1- or RUNX1-IT1-overexpressing PC cells were cotransfected with the WT vector or with one of the MUT vectors. f, g ChIP assays with an anti-RUNX1 antibody or IgG were performed to verify the enrichment of RUNX1 at binding sites in the C-FOS promoter in PC cells. The ChIP-PCR products of the RUNX1, input and IgG groups were detected by agarose gel electrophoresis. h RUNX1-overexpressing PC cells were transfected with RUNX1-IT1 or Control Silencer. Luciferase activity assays were performed. i ChIP assays were performed in RUNX1-IT1 knockdown PC and control cells with an anti-RUNX1 antibody or IgG. j Retrieval of RNA by ChIRP with TERC probes (positive control). ChIRP was performed using PANC-1 cells and TERC lncRNA even, odd or NC (LacZ) probe sets. Purified RNA was then analyzed by qRT-PCR using RNA-positive control primers (TERC) and RNA-negative control primers (GAPDH). k Successful DNA binding by ChIRP with TERC probes. Purified DNA was then analyzed by qRT-PCR using the WNT-1 precursor (positive target) and GAPDH D2 coding region (negative target). l Successful retrieval of RNA by ChIRP with RUNX1-IT1 probes. m DNA binding by ChIRP with the RUNX1-IT1 probe. Purified DNA was analyzed by qRT-PCR using primers specific for the FOS promoter binding region and the GAPDH D2 coding region. n A schematic model of the mechanism underlying the role of RUNX1-IT1 in the progression of PC (ns no significance, *P < 0.05, **P < 0.01, ***P < 0.001).