Butyrate induces profound changes in gene expression related to multiple signal pathways in bovine kidney epithelial cells

Abstract

Background: Global gene expression profiles of bovine kidney epithelial cells regulated by sodium butyrate were investigated with high-density oligonucleotide microarrays. The bovine microarray with 86,191 distinct 60mer oligonucleotides, each with 4 replicates, was designed and produced with Maskless Array Synthesizer technology. These oligonucleotides represent approximately 45,383 unique cattle sequences.

Results: 450 genes significantly regulated by butyrate with a median False Discovery Rate (FDR) = 0 % were identified. The majority of these genes were repressed by butyrate and associated with cell cycle control. The expression levels of 30 selected genes identified by the microarray were confirmed using real-time PCR. The results from real-time PCR positively correlated (R = 0.867) with the results from the microarray.

Conclusion: This study presented the genes related to multiple signal pathways such as cell cycle control and apoptosis. The profound changes in gene expression elucidate the molecular basis for the pleiotropic effects of butyrate on biological processes. These findings enable better recognition of the full range of beneficial roles butyrate may play during cattle energy metabolism, cell growth and proliferation, and possibly in fighting gastrointestinal pathogens.

Figure 1

Cell cycle progressions determined by flow cytometry. Histogram plot of flow cytometry analysis of A: control cells; B: the cells treated by 10 mM sodium butyrate. Deconvolution of the DNA histogram using automated cell cycle analysis with 'Cylchred' software of C: control cells; and D: the cells treated by 10 mM sodium butyrate. Insert: Mean ± SEM (n = 3 per treatment; **: P < 0.01. G1/G0: cells in G1/G0 cell cycle phases; S: cells in S cell cycle phase; M/G2: cells in M/G2 cell Cycle phases). 2C and 4C: two and four copies of DNA content respectively.

Figure 2

Butyrate induced hyperacetylation of histone 3 (H3). This figure is representative of three experiments (n = 3). A: Protein from different samples was separated by SDS PAGE on two identical 4 to 20% polyacrylamide gradient gels. One gel was stained with SimpleBlue (Invitrogen) and one was transferred to a membrane for Western blotting with monoclonal anti- acetyl-phospho H3 and anti- acetyl H3 antibodies; B: Western Blots from three experiments were quantified with NIH Image software. The relative densities were measured and corrected with the protein density. H3: histone 3. **: P < 0.01.

Figure 3

Correlation between levels of gene expression measured by microarray and real-time RT-PCR. The R = 0.867 (R² = 0.76) was calculated using log-transformed values of the fold change obtained for the 30 selected genes (n = 60).