Profiling of Target Genes that Regulate EP-Induced Growth Inhibition of H460 through the Transcriptional and Proteomic Analysis

Abstract

Mechanical forces from outside the cells such as compression, stretching or shear stress are well known to participate in various cellular processes. Elevated pressure (EP) is an extrinsic mechanical force which induces mild hyperoxia under 2 ATA of pressure when applied to cells or whole tissues. To evaluate the effect of EP, we tested cell growth in H460 non-small lung carcinoma cells during 48 hours. Cell growth was inhibited over 25% without DNA damage and cell death, and colony formation was also suppressed at EP compared to cells cultured in conventional cell incubator.To identify target molecules that induces cell growth inhibition, we performed transcriptional and phospho-proteomic analysis with phospho-MAPK array. By transcriptome analysis using RNA-sequencing, we identified that 545 genes has increased and 304 genes has decreased over 2 folds in cells under EP. Many genes were related with membrane proteins, ECM molecules and cytoskeleton molecules. Among them we confirmed the gene including IGFBP5, P2RY6 and IL1R2 by real-time PCR. Among MAPK proteins only Erk phosphorylation was decreased, but not Akt, JNK and MKK3/6 under the exposure of EP for 4 hours. Phospho-proteomic analysis with 2D and PMF (Peptide mass fingerprint) showed increased phosphorylation of 41 proteins including GNB2L1 (Guanine Nucleotide Binding Protein) and decreased phosphorylation of 14 proteins including HELLS (helicase, lymphoid-specific) which are known to regulate cell growth and Erk activation.Our results suggested that EP inhibits cell growth of H460 cancer cells through regulation of various molecules related with cell growth including Erk, IGFBP and GNB2L1.(Medical Research Center for Environmental Toxico-Genomics and Proteomics Project, No.20090091416).