Osteosarcoma is the most common primary bone tumor generally affecting young

Osteosarcoma is the most common primary bone tumor generally affecting young people. thought to be risk factors for developing osteosarcoma the etiology has not been fully understood [1 2 3 Prostaglandin endoperoxide synthase 2 (PTGS2) also called as cyclooxygenase-2 (COX-2) catalyzes the convertsion of arachidonic acid to prostaglandin H2 from which various prostanoids including prostaglandin E2 are produced [4]. Accumulating evidence indicates that COX-2 is involved in osteosarcoma development and progression. Several studies have reported that high levels of COX-2 expression is associated with advanced clinical stage and metastasis [5 6 as well as with lower overall survival rates and disease-free survival rates [7 8 9 In addition COX-2 inhibition by using RNAi or antisense oligonucleotide inhibits cell proliferation and invasion in human osteosarcoma cells [10 11 Also selective COX-2 inhibitors reduce not only osteosarcoma Febuxostat cell proliferation and invasion but also tumor growth and metastasis in vivo [12 13 Moreover we have Febuxostat previously reported that COX-2 overexpression promotes cell proliferation migration and invasion in U2OS human osteosarcoma cells [14]. These studies strongly CACH6 suggest that COX-2 might be a causal factor for the development and progression of osteosarcoma. However the exact mechanisms of action of COX-2 in osteosarcoma are largely unknown. In an attempt to figure out the mechanism of action of COX-2 in osteosarcoma we analyzed the gene expression profiles in three COX-2-overexpressed U2OS stable cell lines and three control Febuxostat stable cell lines. Methods Establishment and maintenance of stable cell lines Human COX-2 cDNA was subcloned into the pcDNA3 vector containing neor. U2OS cells were transfected with COX-2 or pcDNA3 DNA using Lipofectamine2000 (Life Technologies Grand Island NY USA). Transfectants were selected in the presence of geneticin and individual clones were maintained in Dulbecco’s modified Eagle’s medium containing fetal bovine serum (10%) penicillin (100 units/mL) streptomycin (100 units/mL) and geneticin (700 μg/mL) as reported previously [14]. RNA isolation Total RNA was extracted from cells with Trizol (Life Technologies) purified with the addition of chloroform and precipitated with the addition of isopropanol. The RNA concentration was determined by a spectrophotometer and the quality of RNA was evaluated by the OD 260/280 ratio and gel electrophoresis. Hybridization to expression arrays The following procedures were carried out by Macrogen Co. (Seoul Korea). First total RNA was amplified and Febuxostat purified using the Ambion Illumina RNA amplification kit to yield biotinylated cRNA (Ambion Austin TX USA). Briefly 550 ng of total RNA was reverse-transcribed to cDNA using a T7 oligo(dT) primer. Second-strand cDNA was synthesized in vitro-transcribed and labeled with biotin-NTP. After purification 750 ng of labeled cRNA was hybridized to the humanHT-12 expression v.4 bead array (Illumina San Diego CA USA) for 16-18 h at 58℃. The array signal was detected using Amersham fluorolink streptavidin-Cy3 (GE Healthcare Bio-Sciences Little Chalfont UK). Arrays were scanned with an Illumina bead array reader/confocal scanner. Array data were filtered by a detection p-value < 0.05 (similar to signal to noise). Selected gene signal values were log-transformed and normalized by the quantile method. Statistical analysis Basic statistical analyses were performed using Microsoft Excel. Hierarchical cluster analysis was conducted with normalized log2-gene expression values using Cluster 3.0 and the results were visualized using Java Treeview [15 16 An unrooted tree was drawn with R package. Biological function analysis was performed with official gene names using DAVID (http://david.abcc.ncifcrf.gov/). Results Stable cell lines We have previously established stable cell lines over-expressing human COX-2 in U2OS human osteosarcoma cells. To avoid clonal variations we established three stable COX-2-overexpressing cell lines (U2OS-COX-2.