Simultaneous gene expression analysis of steady-state and actively translated mRNA populations from osteosarcoma MG-63 cells in response to IL-1alpha via an open expression analysis platform

Abstract

Pro-inflammatory cytokines play a key role in various forms of metabolic bone diseases, including osteopenia and osteoporosis. Human MG-63 cells treated with IL-1alpha were used as a model system to identify potential marker genes that are differentially expressed. This study is designed to quantitate gene expression of actively translated mRNAs as compared to the steady-state mRNA population. Both steady-state mRNAs and actively translated mRNAs from control MG-63 cells and MG-63 cells treated with IL-1alpha were isolated and converted to cDNA. The gene expression analysis from these samples was then quantitated with an open expression analysis platform with no requirement for a priori knowledge of sequence information. As a result, many differentially regulated genes were discovered via IL-1alpha treatment. Some of the genes have been described previously as playing important roles in the regulation of inflammation and cell adhesion. These comparisons provided a panoramic overview of gene expression at both the total transcript and post-transcriptional levels. In addition, the quantitation of actively translated mRNAs associated with polysomes also provided a better estimation of protein expression levels. This methodology allows for the identification of genes acutely regulated during translation. Furthermore, the process may aid in the identification of new drug targets or biomarkers.

Figure 1

Schematic diagram of polysomal sample preparation and quantitative expression analysis (GeneCalling). Details are described briefly in Materials and Methods (20).

Figure 2

Polysome distribution of cellular mRNAs (n = 3). Polysomal distribution of MG-63 control cells and cells treated with IL-1α for 6 h. Absorbance profiles at 254 nm of the collected sucrose gradients are shown. Fractions 1–7 represent free mRNA and monosomes; fractions 8–13 contain the polysomes.

Figure 3

GeneCalling analysis of cell cycle-related genes with Spotfire software. The fold changes (P < 0.01, fold changes > 2) in the mean values (n = 3) of these genes in steady-state GeneCalling from control MG-63 cells and MG-63 cells treated with IL-1α were compared with the mean values (n = 3) of the corresponding genes in actively translated GeneCalling analysis. Both actively translated and steady-state samples were compared. Job22012, actively translated GeneCalling analysis; Job22075, steady-state mRNA GeneCalling analysis.

Figure 4

Up-regulation of human PP-2A expression by IL-1α treatment. (A) Trace replication of QEA electrophoresis output for PP-2A from total mRNA of MG-63 control cells (Set B) and cells treated with IL-1α (Set A). (B) Trace replication of QEA electrophoresis output for PP-2A from polysomal isolated mRNA of MG-63 control cells (Set B) and cells treated with IL-1α (Set A). Red line at peak position 307.6 indicates the gene fragment corresponding to PP-2A.

Figure 5

(A) RT–QPCR analysis of PP-2A expression in MG-63 cells. The average CT values for PP-2A were first normalized against the housekeeping gene GAPDH and converted to a percentage for relative expression (n = 3). (B) Western immunoblot analysis of PP-2A in MG-63 cells. Cytosolic extracts from MG-63 cells (lane 1) and MG-63 cells treated with IL-1α (lane 2) were prepared. PP-2A protein was detected using an anti-PP-2A mouse monoclonal antibody. Filtered membranes were then reprobed with an anti-β-actin monoclonal antibody to control for loading and integrity of protein.

Figure 6

(A) Trace replication (n = 3) of QEA electrophoresis output for translation initiation factor 4B from steady-state mRNA of MG-63 cells (Set B) and cells treated with IL-1α (Set A). (B) Poisoned QEA electrophoresis output from isolated polysome mRNA of MG-63 cells (Set B) and cells treated with IL-1α (Set A). Red line at peak position 358.0 indicates gene fragment corresponding to translation initiation factor 4B.

Figure 7

(A) RT–QPCR analysis of CAML expression in MG-63 cells. CT values for CAML were first normalized against housekeeping gene GAPDH and converted to a percentage for relative expression. (B) Western immunoblot analysis of CAML in MG-63 cells. Cytosolic extracts from MG-63 cells (lane 2) and MG-63 cells treated with IL-1α (lane 1) were prepared. CAML protein was detected by immunoblot analysis using an anti-CAML polyclonal antibody. Filtered membranes were then reprobed with an anti-β-actin monoclonal antibody to control for loading and integrity of protein.