Identification of tumor invasion-related differentially expressed genes in different grades and all-trans retinoic acid-treated astrocytoma cell lines

摘要

BACKGROUND:Although several genetic aberrations and gene expressional changes have been shown to exist in tumors and different grades of astrocytomas,as well as in normal tissues,the gene profiling and ge-netic pathways associated with malignant transformation and progression remain unclear. OBJECTIVE: To identify differentially expressed genes related to tumor invasion from various grades and all-trans retinoic acid (ATRA)-treated astrocytoma cell lines by cDNA microarray. DESIGN,TIME AND SETTING: In vitro gene experiment was performed at the Department of Neurobi-ology,Third Military Medical University of Chinese PLA from January to October 2007. MATERIALS: Two different grades of astrocytoma cell lines CHG-5 (WHO grade Ⅱ) and SHG-44 (WHO grade Ⅳ) were developed by our laboratory; a cell differentiation-inducing agent ATRA and a human cDNA microarray technology were used to determine differentially expressed genes (City University of Hong Kong). METHODS: Total RNA was extracted using the Trizol test kit. Reverse transcription was performed using Superscript Ⅱ reverse transcriptase. The cDNA product (target DNA) was marked with fluorochromes Cy3 (normal SHG-44) and Cy5 (CHG-5 or ATRA-treated SHG-44),followed by chip hybridization. MAIN OUTCOME MEASURES: Gene expression profiles of CHG-5 vs. SHG-44 and ATRA-treated vs. normal SHG-44 were performed to identify differentially expressed genes. Several of these genes were ran-domly selected for Northern Blot analysis. The identification of genes that were similarly regulated (overlap-ping) was performed by comparing gene expression profiles between CHG-5 and SHG-44 cells,and between SHG-44 cells with or without treatment with ATRA. RESULTS: No significant differences were observed between CHG5 and SHG-44 cell line morphology. Under confocal microscopy,GFAP staining intensity of CHG5 cells was greater than SHG-44 cells (t=6.078,P=0.004). Growth curve analysis demonstrated that the speed of SHG-44 cell growth was greater than CHG5 cells. Flow cytometry analysis showed that the number of ATRA-treated SHG-44 cells at G0/G1 stage increased by 15%,compared with normal SHG-44 cells (P<0.05). A total of 31 known genes with altered expression were identified in this study. Among them,20 genes were upregulated and 11 were downregulated in CHG-5 compared with SHG-44 cells,and ATRA-treated SHG-44 compared with untreated SHG-44 cells. Four of these reported genes (CD151,G3BP,UGB,and CSTB) were shown to be involved in tumor invasion. Validation of a selection of differentially expressed genes was performed by Northern blot. CONCLUSION: A total of 31 known genes were demonstrated by cDNA microarray to relate to the malig-nant progression of astrocytomas,and four differentially expressed genes (CD151,G3BP,UGB,and CSTB) were shown to relate to tumor invasion.