The S100A9/NF-κB/SAA3/miR-204Loop In Mouse Macrophage Affects The Colitis-associated Carcinogenesis

Nonresolving inflammation is associated with tumorigenesis and progression. The incidence of colorectal cancer is rising at home and abroad. It is one of the most common types of cancer and causes about655000deaths per year worldwide. Because of the nonresolving colitis associated colorectal cancer has a very clear inflammatory background, and this background affect the pathogenesis a lot, so the mechanism of nonresolving colitis associated colorectal cancer is different from classic colorectal cancer. The pathogenesis of NCAC is still not clear. During the previous study, we establish a NCAC mouse model, which contain an inflammatory-dysplasia-cancer dynamic progression. We analysis the dynamic transcriptome of the whole disease, and find the key points of the regulation network. The results showed that, the expression level of SAA3, S100A8and S100A9sustained high level during the whole develop progress. NF-κB, MAPK, Wnt/(3-catenin signaling pathway play a very important role in NCAC. So we chose SAA3, S100A8and S100A9as a starting point to further study.The RNA was extracted from the intestinal mucosa and the qRT-PCR was putted to detect the expression of SAA3, S100A8and S100A9at the gene level. Another part of the mucosa produced tissue sections, and immunohistochemistry was used to detect the expression patterns of SAA3, S100A8and S100A9at protein levels. The serum was collected to test serum SAA3, S100A8, S100A9expression using ELISAassay. These three molecules all up regulated in inflammatory cell and the epithelial cells which with malignant transformation. We used macrophages as experimental subjects to represent the inflammatory microenvironment. Exogenous S100A8, S100A9recombinant protein stimulated macrophages, and then the expression level of endogenous SAA3was increased by using qRT-PCR detection. Western blot was used to detect the expression of p-AKT increased, while the IκB expression decreased. Furthermore we used the Dual-Luciferase Reporter Assay System to test the activation of NF-κB, the results showed that exogenous S100A8and S100A9activated NF-kappaB and PI3K/AKT signaling pathway in the macrophages, and prompt SAA3up-regulated by activation these two signaling pathway. We use bioinformatics methods predicted the microRNA which can regulate the expression of SAA3, S100A8and S100A9at post-transcript level. And the results showed that microRNA204may be regulating the expression of S100A9. When use the SAA3over expression vector transiently transfected macrophages resulting in SAA3over-expressed, we found endogenous S100A9expression increases while microRNA204expression down-regulated. Furthur, we cloned the DNA sequence which with the wild type and mutant type of microRNA204binding site in S100A93’-UTR into pMir-Report plasmid, respectively. microRNA204expression vector were co-transfected into293T cells, and the luciferase activity was determined. The luciferase reporter assay showed microRNA204reduced the luciferase activity of the vector with wild type S100A93’-UTR, suggesting microRNA204directly targeted S100A9.We found ectopic expression of SAA3and conditioned culture median from macrophage RAW264.7cell stimulated with recombination protein S100A8or S100A9significantly promoted CT26colon cancer cells growth determined with colony formation assay. Transwell assay and MTS assay showed SAA3also promoted CT26invasion and resistance to Oxaliplatin. In regard to the mechanism, we found SAA3overexpression leaded to the location of β-catenin to nuclear, which would be responsible for the upregulation of CCND2, MMP9and Bcl-2, associated with cell invasion and cell survival. In fact, knockdown of β-catenin with sh-RNA in did inhibited cell invasion and reversed the resistance to oxaliplatin. In addition, as a negative regulator of β-catenin, GSK3β was downregulated in SAA3overexpression CT26cells, and re-expression of GSK3β showed the same effect of β-catenin knockdown. Furthermore, ERK was activated in SAA3overexpression CT26cells and ERK inhibition with specific inhibitor restored GSK3β expression and sensitivity to oxaliplatin, with downregulation of CCND2, MMP9and BCL-2.Collectively, in tumors, the infiltrative inflammatory cells such as macrophage, S100A8and S100A9upregulated SAA3expression via activation of PI3K/AKT/NF-κB signal pathway, then SAA3enhanced S100A9expression by inhibiting miR-204expression that directly targeted to S100A9. The regulatory loop in inflammatory cells maintains the high expression of SAA3in the progression of inflammation-associated cancer. In cancer, SAA3triggered ERK/GSK3β/β-catenin pathway to support cancer progression with more proliferation ability, invasive potential and resistance to chemotherapy.