Title: Activation of the Tumor Suppressor MicroRNA-29c by the Selective Cyclooxygenase-2 Inhibitor, Celecoxib in Human Gastric Cancer
Abstract: Background and Aims: IBS is a complex disease with recurring clusters of symptoms.Genetic mutations and single nucleotide polymorphisms do not fit these traits.We reported previously that neonatal inflammatory insult enhances smooth muscle reactivity to ACh in adulthood, which enhances colonic motor function to induce diarrhea-like conditions.In this study, we tested the hypothesis that adverse early life events reprogram the expression of genes encoding vasoactive intestinal polypeptide (VIP) and pore-forming α 1C -subunit of the Ca v 1.2b channels (Canca1c) by epigenetic mechanisms in colonic muscularis externa to induce colonic motility dysfunction in adulthood.Methods: Ten-day old rat neonates received intraluminal 130 mg/kg TNBS in the colon; control pups received saline.Colonic muscularis externae (ME) were obtained 6 to 8-week later.Results: qRT-PCR and western blotting showed significant increases in the mRNA and protein expressions of VIP and α 1C -subunit in the ME of about 50% of adult rats that received neonatal inflammatory insult (IBS-rats).Chromatin immunoprecipitation assays showed marked increase in acetylation of histone H3 lysine 9 (H3K9Ac, a marker of transcriptional activation) (0.8±0.1 vs. 0.45±0.06Ctr., p<0.05) and attenuation in trimethylation of histone H3 lysine 9 (H3K9me3, a marker of transcriptional repression) (0.8±0.07 vs.1.2±0.03Ctr., p<0.05) in the core promoter region of Cacna1c in IBS-rats.As a result, the RNA polymerase II (RNAP II) association with the Cacna1c promoter significantly increased, indicating enhanced Cacna1c gene transcription.We investigated the role of VIP in the epigenetic modification of Cacna1c by incubating ME from naïve rats with 100 ng/ml VIP for 24 hours.VIP-treatment markedly increased H3K9Ac and decreased H3K9me3 at the upstream CREB-binding site (-2062/-2055) and around the transcription start site (+21 ~+168), and hence enhanced RNAP II interaction with this promoter region suggesting that VIP modulates epigenetic components at the Cacna1c promoter to stimulate Cacna1c gene transcription.VIP antagonist, (p-Chloro-D-Phe6,Leu17)-VIP, abrogated VIP-mediated RNAP II binding, H3K9 acetylation and trimethylation at the Cacna1c promoter.Intraperitoneal administration of 100 mM sodium butyrate, a histone deacetylase inhibitor, significantly elevated Cacna1c mRNA in ME of naïve adult rats (1.5±0.06 vs. 1±.01Ctr., p<0.05), indicating that histone hyperacetylation is necessary for α 1C gene transcription.Conclusions: Our findings reveal the epigenetic mechanisms by which adverse early life events alter the gene expression of specific cell signaling proteins to cause colonic motor dysfunction in adulthood.The intensities of epigenetic modifications are modifiable by environmental influences, such as stress, which explains relapses and remissions of symptoms in IBS patients.