Transcriptional network analysis applied to the regulation of 1-a-hydroxylase in macrophages challenged by LPS

MARTINELLI, R.; DAURELIO, L.D.; ESTEBAN, L.

San Pablo

Congreso; X-Meeting 2015-11th International Conference of the AB3C + Brazilian Symposium of Bioinformatics; 2015

The Brazilian Association of Bioinformatics and Computational Biology (AB3C)

The vitamin D can be activated by the rate-limiting enzyme 1-a-hydroxylase (CYP27B1) synthesized mainly by renal cells. But other kind of cells can produce it and its regulation has different characteristics. In cells of the immune system, this enzyme is under the control of immune stimuli. For example, macrophage exposure to lipopolysacharide (LPS) has been implicated in the up-regulation of CYP27B1. The aim of this work is, by means of a system approach, to examine the complex interaction that involves the enzyme regulation in macrophages challenge with LPS. The transcription profiles of GSE40885 (Affymetrix Human Genome U133 Plus 2.0 Array) were downloaded from Gene Expression Omnibus (GEO). In this experiment, the RNA was isolated from alveolar macrophages of healthy men (n=14) by bronchoscope after the instillation of sterile saline or lipopolysacharide (LPS), and were divided into exposure group and control group accordingly. The raw data was preprocessed (correction, normalization) by RMA function of affy package of R 3.03 software in Bioconductor (http://www.bioconductor.org/). We applied Weighted Gene Co-expression Network Analysis (WGCNA) to detect clusters of highly co-expressed genes (modules). The weighted adjacency matrix assesses continuous connection strength ([0, 1]) according to b parameter for each group. Adhering to the scale-free topology criterion, a=6 was considered. The co-expression matrix and the topological overlap matrix (TOM) were constructed subsequently. With average linkage hierarchical clustering, gene modules were identified for each group. WGCNA analysis assesses the correlation between genes and considers degree of gene shared neighbors across the whole network. Moreover, WGCNA can provide connection strength between the genes, unlike general co-expression network. The module containing CYP27B1 was identified, and co-expression with c/EBPb and NfkB, both related with the 1-a-hydroxylase gene regulation, was found. Enrichment in Pathways using DAVID (https://david.ncifcrf.gov/) was done to get functionality information about this module. The gene list was enriched in 17 KEGG pathways, from which "p53 signaling pathway" and "Pancreatic cancer" were the most statistically significant. In addition, it was involved in the "Cell cycle" pathway. These biological activities are related to the well-known vitamin D anti-proliferative actions and result interesting to to find potential therapeutic targets.