Bacteria use quorum-sensing molecules (QSMs) to communicate within as well as across species. However, the effects of QSMs on eukaryotic host cells have received limited attention. We report that the quorum-sensing pentapeptide, competence and sporulation factor (CSF), of the Gram-positive bacterium Bacillus subtilis activates key survival pathways, including p38 MAP kinase and protein kinase B (Akt), in intestinal epithelial cells. CSF also induces cytoprotective heat shock proteins (Hsps), which prevent oxidant-induced intestinal epithelial cell injury and loss of barrier function. These effects of CSF depend on its uptake by an apical membrane organic cation transporter-2 (OCTN2). Thus, OCTN2-mediated CSF transport serves as an example of a host-bacterial interaction that allows the host to monitor and respond to changes in the behavior or composition of colonic flora.

Colonic microbiota ferment non-absorbed dietary fiber to produce prodigious amounts of short chain fatty acids (SCFAs) that benefit the host through a myriad of metabolic, trophic, and chemopreventative effects. The chemopreventative effects of the SCFA butyrate are, in part, mediated through induction of p21 gene expression. In this study, we assessed the role of microRNA(miRNA) in butyrate's induction of p21 expression. The expression profiles of miRNAs in HCT-116 cells and in human sporadic colon cancers were assessed by microarray and quantitative PCR. Regulation of p21 gene expression by miR-106b was assessed by 3′ UTR luciferase reporter assays and transfection of specific miRNA mimics. Butyrate changed the expression of 44 miRNAs in HCT-116 cells, many of which were aberrantly expressed in colon cancer tissues. Members of the miR-106b family were decreased in the former and increased in the latter. Butyrate-induced p21 protein expression was dampened by treatment with a miR-106b mimic. Mutated p21 3′UTR-reporter constructs expressed in HCT-116 cells confirmed direct miR-106b targeting. Butyrate decreased HCT-116 proliferation, an effect reversed with the addition of the miR-106b mimic. We conclude that microbe-derived SCFAs regulate host gene expression involved in intestinal homeostasis as well as carcinogenesis through modulation of miRNAs.

BackgroundCompromised colonic butyrate production resulting from low dietary fiber or altered gut microbiota may promote colon neoplasia. Previous reports indicate these actions are mediated in part by altered levels of miRNAs, including suppressed expression of the oncogenic miR-17-92a cluster. Here, we sought to identify the mechanisms underlying these effects of butyrate in colon cancer.MethodsmiR-92a levels were measured in archived human colon cancer and adjacent normal colon specimens by microarray and quantitative RT-PCR (qPCR). The effects of butyrate and other histone deacetylase inhibitors (suberoylanilide hydroxamic acid (SAHA) and valproic acid) on primary (pri-miR17-92a), precursor and mature miR-92a were analyzed in HCT-116 and HT-29 human colon cancer cells using qPCR. The effects of butyrate, SAHA and valproic acid on protein levels of c-Myc, Drosha and p57 were measured in HCT-116 cells using immunoblotting. Regulation of C13orf25 promoter activity by butyrate was analyzed by luciferase reporter assay using modified pGL3 constructs containing a wild-type or mutated c-Myc binding site. Expression of c-Myc was modulated using siRNA or adenovirus vectors. p57 mRNA and protein were measured before and after transfection with miR-92a-mimic molecules. Following butyrate treatment and miR-92a-mimic transfection, apoptosis was analyzed by TUNEL staining and caspase-3 immunoblotting.ResultsMicroarray, confirmed by qPCR, revealed a seven-fold increase in miR-92a levels in sporadic human colon cancer tissue compared to adjacent normal colon. Treating human colon cancer cells with butyrate reduced the levels of pri-miR17-92a, precursor and mature miR-92a, as well as c-Myc. SAHA and valproic acid had similar effects. Mutation of the c-Myc binding site diminished butyrate’s inhibitory effects on C13orf25 promoter activity. Silencing c-Myc expression reduced miR-92a levels. c-Myc over-expression neutralized butyrate-induced attenuation of pri-miR17-92a. Exogenous miR-92a inhibited butyrate-induced p57 expression and reversed the beneficial actions of butyrate on colon cancer cell proliferation and apoptosis.ConclusionsOur findings identify a novel cellular mechanism whereby butyrate inhibits miR-92a transcription by reducing c-Myc, thus augmenting p57 levels. These actions diminish colon cancer cell proliferation and stimulate apoptosis. This newly described regulation of oncogenic miRNA biogenesis expands our understanding of colon cancer cell biology and identifies novel therapeutic targets.

Background & Aims-Inducible heat shock proteins (iHsp), Hsp25/27 and Hsp70, play essential roles in protecting cells against stress and, in intestinal mucosal inflammation, potentially lessening the extent and severity of injury. We examined the expression and regulation of iHsp in human and experimental inflammatory bowel diseases (IBD) and in vitro.