We have previously demonstrated that neuropeptide Y (NPY), a peptide neurotransmitter, abundantly produced by enteric neurons, is an important regulator of intestinal inflammation, and that NPY knockout mice exhibit attenuated inflammation. However the role of NPY in the progression of chronic inflammation to tumorigenesis is unknown. We investigated whether NPY could modulate epithelial cell proliferation and apoptosis, and thus regulate tumorigenesis.Methods:
Repeated cycles of dextran sodium sulfate (DSS, comprising of 3 cycles of 1 week of 3% DSS alternating with 2 weeks of regular water cycle) was used to model inflammation-induced tumorigenesis in wild-type (WT) and NPY knockout (NPY−/−) mice. Intestinal epithelial cell lines (T84) were used to assess the effects of NPY (0.1 μM) on epithelial proliferation and apoptosis in vitro.Results:
We observed that DSS-WT mice exhibited enhanced intestinal inflammation, polyp size and polyp number (7.5 ± 0.8) compared to DSS-NPY−/− mice (4 ± 0.5, P < 0.01). Accordingly, DSS-WT mice also showed increased colonic epithelial proliferation (PCNA, Ki67) and reduced apoptosis (TUNEL) compared to DSS- NPY−/− mice (P < 0.05). The apoptosis regulating microRNA, miR-375, was significantly down regulated in the colon of DSS-WT (2-fold, P < 0.01) compared to DSS-NPY−/− -mice. In vitro studies indicated that NPY promotes cell proliferation (increase in PCNA and β-catenin, P < 0.05) via phosphatidyl-inositol-3-kinase (PI3-K)—β-catenin signaling, suppressed miR-375 expression and reduced apoptosis (increase in phospho-Bad). NPY-treated cells also displayed increased c-myc and cyclin D1, and reduction in p21 (P < 0.05). Addition of miR-375 inhibitor to cells already treated with NPY did not further enhance the effects induced by NPY alone.Conclusions:
Our findings demonstrate a novel regulation of inflammation-induced tumorigenesis by NPY-epithelial cross talk as mediated by activation of PI3-K signaling and down regulation of miR-375. Our data unravels a novel role of NPY in regulating inflammation-induced tumorigenesis via 2 modalities -by enhanced proliferation (PI3-K/pAkt), and secondly by downregulation of microRNA-375 (miR-375)-dependent apoptosis in intestinal epithelial cells. Our data establishes the existence of a microRNA-mediated cross talk between enteric neurons producing NPY and intestinal epithelial cells, and the potential of neuropeptide-regulated miRNA's as potential therapeutic molecules for the management of inflammation-associated tumors in the gut.