The Microbe-Derived Short Chain Fatty Acid Butyrate Targets miRNA-Dependent p21 Gene Expression in Human Colon Cancer

Abstract

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.

Influence of miRNA-106b and miRNA-135a on butyrate-regulated expression of p21 and Cyclin D2 in human colon adenoma cells

“…A validated target gene of miR-106b is the cell cycle regulator p21. MiR-106b binds to the 3'UTR and inhibits the translation of p21 (Hu et al 2011). In the present study, butyrate and TSA were able to reduce miR106b expression in LT97 colon adenoma cells, especially after 48-h treatment and in HT29 cells (Supplementary Figure S1).…”

“…Butyrate regulates the expression of genes and proteins involved in cell cycle control like p21 and Cyclin D1, leading to a cell cycle arrest in human colon cancer cells (Hamer et al 2008;Hinnebusch et al 2002;Siavoshian et al 2000). Lately, Hu et al (2011) as well as Humphreys et al (2013) showed that butyrate also influences miRNA expression in HCT116 and HT29 colon cancer cells, respectively. Therefore, it can act both via its function as HDI and by regulation of miRNA which are dysregulated in CRC (Bartley et al 2011;Schetter et al 2008;Slattery 2000;Wu et al 2011).…”

“…Therefore, in the present study we investigated for the first time the influence of butyrate on the expression of proliferation and cell cycle-relevant miRNAs (miR-135a, miR-135b, miR-24, miR-106b, miR-let-7a) in LT97 colon adenoma cells as an early stage of transformed cells. The HDI TSA was used as a positive control (Hu et al 2011;Kiefer et al 2006). All analyzed miRNAs were down-regulated to a certain degree by butyrate or TSA after 24-or 48-h incubation, respectively.…”

“…The expression of miR-106b was significantly reduced upon treatment with both HDIs butyrate and TSA, especially after 48 h. Several studies have shown that miR106b is overexpressed in colon cancer cells and that it has oncogenic potential (Hu et al 2011;Schetter et al 2008;Wu et al 2011). A validated target gene of miR-106b is the cell cycle regulator p21.…”

“…In contrast, it could be shown that butyrate functions as histone deacetylase inhibitor (HDI), inhibits proliferation and induces apoptosis and differentiation in colon cancer cells mediated, e.g., by induction of p21 gene expression (Borowicki et al 2010;Hinnebusch et al 2002). There is evidence from two studies that these chemopreventive effects of butyrate are mediated also by miRNAs since butyrate as HDI also regulates miRNA expression profiles in colon cancer cells (Hu et al 2011;Humphreys et al 2013). Until now, the role of miRNAs in butyratemediated chemopreventive effects on colon adenoma cells at an early stage of colon cancer development has not been investigated yet.…”