Identification Of Differentially Expressed Genes In MG63 Drug-resistant Human Osteosarcoma Cells By Microarray Analysis

Objective: The differential expression of genes in osteosarcoma resistant cells and their primary cells was compared by gene chip,and the results of chip verification were established,which lay the foundation for the development of target genes in clinical diagnosis of osteosarcoma..Methods: 1.The sensitivity of human osteosarcoma MG63 cells and vincristine-resistant MG63 cells(MG63/VCRcells)for vincristine were detected by the CCK-8 assay.2.High-quality total RNA which isolated from MG63/VCR cells and MG63 cells were used for microarray analysis;the m RNA in the total RNA is amplified,labeled and purified by Microarray kit Gene Chip3’IVT Express Kit of Affymetrix;According to Gene Chip Hybridization,Wash and Stain Kit reagent,hybrid reaction fluid and chip were prepared for hybridization and washing;By scanning and reading raw data,the results of the chip were obtained,and the gene of difference expression was selected after the quality control was processed.3.Results were analyzed bioinformatically to predict their roles in the development of drug resistance by using Ingenuity Pathway Analysis(IPA) online software.Quantitative reverse-transcription polymerase chain reaction(q RT-PCR)was employed tovalidatethe reliability of the gene chip r esults.Results: 1.Comparison of the median inhibitory concentration of vincristine IC50,multidrug resistant cells of osteosarcoma MG63/VCR is 493.175±4.473 ng/ml,MG63 is 1.407±0.111 ng/ml(p<0.01)2.A total of 1300 genes(602 upregulated and 698 downregulated)were differentially expressed in the MG63/VCR cells compared with MG63 cells,according to the microarray results.45 downregulated genes and 26 upregulated genes that were statistically significant with a p value < 0.05 as well as expressed at four-fold or greater difference between the two cells were selected for further analysis,there were 4 transcriptional factors,12 enzymes,5 transporters,4 transmembrane receptors,and 46 other.3.Results were analyzed bioinformatically to predict that the differentially expressed genes were mainly enriched in UVA-Induced MAPK Signaling(PIK3R1,MAPK9 and EGFR etc.),Erb B2-Erb B3 Signaling(RRAS2,STAT5 B,and ATM etc.),these pathways were implicated in regulating organismal death,cell death of immune cells or chemotherapy resistance(P<0.05).10 selected genes(ROBO1,DAPK1,and AKAP12 etc.)were verified by q RT-PCR,which confirmed that the selected genes were abnormal expression between the MG63/VCR cells and MG63 cells,and 90% of which were consistent with the gene chip expression.Conclusions: 1.Compared with MG63 cells,MG63/VCR cells highly resistant to vincristinedemonstrated by Cell viability assay(P<0.01).2.Comparative analysis of gene chip by osteosarcoma cells and drug resistance of osteosarcoma cells,found many new gene expression differences,the validation results show that the difference of the expression of gene chip screening is reliable,including ROBO1,DAPK1,and AKAP12 genes can be used as a new therapeutic target for drug resistant osteosarcoma cells.