Butyrate modulates bacterial adherence on LS174T human colorectal cells by stimulating mucin secretion and MAPK signaling pathway

BACKGROUND/OBJECTIVES: Fermentation of dietary fiber results in production of various short chain fatty acids in the colon. In particular, butyrate is reported to regulate the physical and functional integrity of the normal colonic mucosa by altering mucin gene expression or the number of goblet cells. The objective of this study was to investigate whether butyrate modulates mucin secretion in LS174T human colorectal cells, thereby influencing the adhesion of probiotics such as Lactobacillus and Bifidobacterium strains and subsequently inhibiting pathogenic bacteria such as E. coli. In addition, possible signaling pathways involved in mucin gene regulation induced by butyrate treatment were also investigated. MATERIALS/METHODS: Mucin protein content assay and periodic acid-Schiff (PAS) staining were performed in LS174T cells treated with butyrate at various concentrations. Effects of butyrate on the ability of probiotics to adhere to LS174T cells and their competition with E. coli strains were examined. Real time polymerase chain reaction for mucin gene expression and Taqman array 96-well fast plate-based pathway analysis were performed on butyrate-treated LS174T cells. RESULTS: Treatment with butyrate resulted in a dose-dependent increase in mucin protein contents in LS174T cells with peak effects at 6 or 9 mM, which was further confirmed by PAS staining. Increase in mucin protein contents resulted in elevated adherence of probiotics, which subsequently reduced the adherent ability of E. coli. Treatment with butyrate also increased transcriptional levels of MUC3, MUC4, and MUC12, which was accompanied by higher gene expressions of signaling kinases and transcription factors involved in mitogen-activated protein kinase (MAPK) signaling pathways. CONCLUSIONS: Based on our results, butyrate is an effective regulator of modulation of mucin protein production at the transcriptional and translational levels, resulting in changes in the adherence of gut microflora. Butyrate potentially stimulates the MAPK signaling pathway in intestinal cells, which is positively correlated with gut defense.

Ascl2 knockdown in colon cancer LS174T cells led to expression change of EMT-associated microRNA / 重庆医学

Objective To investigate the effects of transcription factor achaete scute-like 2(Ascl2)on epithelial-mesenchymal transition (EM T ) associated microRNAs .Methods Colon cancer LS174T cells were transfected with shRNA-Ascl2 vector and shRNA-control vector respectively ,then the transfected cells were selected with G 418 and stably transfected cell lines were estab-lished .Real-time PCR and Western-blot analysis were used to determine the interference effect .Transwell invasion experiment were used to observe the effects of Ascl2 RNA interference on cell invasion capability in vitro .MicroRNA chip analysis was used to de-tect the change of EMT-associated microRNA expression ,and real-time PCR experiment was used to validate the microarray re-sults .Results The mRNA and protein expressions were significantly reduced after Ascl2 interference (P<0 .01) .The numbers of invading cells were significantly decreased after Ascl2 interference (P<0 .01) .MicroRNA chip analysis found microRNA-200 fami-ly (including microRNA-200b ,microRNA-200a ,microRNA-429 ,microRNA-200c ,microRNA-141) was more than 2-fold upregula-tion after Ascl2 interference (P< 0 .01) .Conclusion Ascl2 regulates the invasion and metastasis of colon cancer cell ,possibly through transcription regulation of microRNA-200 family ,and then regulation of EM T .