Fig. 8

CXCL13/CXCR5/NFκB/p65/miR-934 positive feedback loop mediates the interaction between M2 macrophages and CRC cells during CRLM. a Western blot assays were performed to evaluate the expression levels of total p65, phosphorylated p65, IκBα, MMP2, and MMP9 in SW480 and RKO cells pretransfected with exogenous CXCL13 or shCXCR5. b PMA-treated THP-1 cells were transfected with miR-934 mimics and treated with anti-CXCL13 antibody or transfected with shCXCR5. The CM of these THP-1 cells was added to SW480 and RKO cells, and the expression of p65, phosphorylated p65, IκBα, MMP2, and MMP9 was determined again using western blot assay. c SW480 and RKO cells were transfected with exogenous CXCL13 and cocultured with or without an inhibitor of NFκB/p65 signaling (helenalin), and the expression of miR-934, MMP2, and MMP9 was examined using qPCR. d Schematic diagram representing the two specific binding sites between p65 and the promoter of miR-934. E. A ChIP assay was used to evaluate five potential binding regions between p65 and the miR-934 promoter in SW480 and RKO cells via LASAGNA-Search 2.0 and the human genomic databases of the National Center for Biotechnology Information (NCBI). f Mutants with truncated binding sites and their control vectors were cloned into pGL3-luciferase reporter plasmids and then transfected into SW480 and RKO cells. g A luciferase reporter gene activity assay was used to analyze the effects of helenalin (a p65 inhibitor) as well as the CM of miR-934-overexpressing THP-1 cells (PMA pretreatment) on the luciferase activity of the WT or MUT p65 binding site 1 of miR-934 promoter constructs in SW480 and RKO cells. h qPCR assays were performed to analyze the effects of helenalin on the RNA expression levels of miR-934 in SW480 and RKO cells cocultured with the culture medium of PMA-treated THP-1 cells treated with miR-934. i Graphical illustration of the interaction between TAMs and CRC cells (*p < 0.05; **p < 0.01; ***p < 0.001)