Analysis Of CD44(+)CD24(-)AGS Gastric Cancer Cell Growth And Stem Cell Related Gene Expressions

ObjectiveIn this study, CD44(+) CD24(-) cells was firstly screened from the humangastric cancer AGS cells. Cell growth characteristics was observed including cellproliferation, tumor sphere formation, migration and invasion, and to study themechanisms that involved in maintenance of the stemness of the cancer stem cells.Gene chip analysis was used to analyze gene expression difference between CD44(+)CD24(-) cells and CD44(-) CD24(-) cells, which will help to find stem cells relatedgenes.MethodsWe isolated CD44(+) CD24(-) AGS cells with MACS(magnetic cell sorting)AGS cells, and detected the expression of cell surface molecules and differentiationmarkers by flow cytometry analysis. CD44(+) CD24(-) cell proliferation capacitywere observed with tumor sphere formation assay. Migration and invasion ability ofCD44(+) CD24(-) AGS cell subsets were displayed with migration the invasionassay analysis. The DNA expression level of CD44(+) CD24(-) cells and unsortedAGS cells was detected with microarray that was provided by Roche NimbleGen.analysis found that stem cell-related gene beta-catenin and EpCAM, respectively, byRT-PCR and Western Blot verify. The accuracy of the results of microarray wasjudged by the coincidence of the results and that of RT-PCR and Western blot.Results1. Highly purified CD44+CD24-cells was harvested with MACS.The FCM analysisresults show that in AGS cells, CD44(+) AGS cells accounted for about5.15%,and CD24(+) AGS cells accounted for only0.88%. Sorting using the MACStechnology, the CD44(+) CD24(-) AGS cells, including CD44-positive rate was99.83%, CD24-negative rate of99.9%; 2. The tumor sphere formation efficiency of CD44(+) CD24(-) AGS cells was higherthan that of control unsorted AGS cells (P<0.05).3. Compared with the unsorted AGS cells, the migration and invasion abilities ofCD44+CD24-cells were promoted(P<0.05).4. From DNA microarray analysis, it showed that347differentially expressed genesexpression was obviously different between CD44(+) CD24(-) and unsorted cells,of which199genes up-regulated,148genes down-regulated. find two stemcell-related differences the expression the the gene beta-catenin, EpCAM;5. The mRNA and protein expression of beta-catenin and EpCAM was examined byRT-PCR and Western blot respectively. Both of them in CD44(+) CD24(-) cellsexpressed significantly increasingly compared with unsorted cells(P<0.001).Conclusion1. The CD44(+) CD24(-) AGS cells appear stemness-like biological characteristics.2. Microarray could screen for genes related to gastric cancer stem cells in AGS cellline effectively. There are347different expressed genes totally, includingup-regulated beta-catenin and EpCAM.3. Identification of stem cells related genes, beta-catenin and EpCAM, provides atheoretical basis to discover markers of gastric cancer stem cell.