Screening Of Differentially Expressed Genes In Osteosarcoma MG-63 Side Population Cells By Expression Beadchip And Functional Verification Of Candidate Genes

Objective:Flow cytometry was used to sort the side population cells and non-side population cells of human osteosarcoma cell line MG-63 cells.The human expression beadchip was used to detect the differentially expressed genes between the two cells.After analysis,comparison and verification of differentially expressed genes,candidate genes were identified.Gene overexpression and silencing techniques were used to verify the regulatory relationship between candidate differential genes and the characteristics of tumor stem cells,which provided a theoretical basis for the discovery of therapeutic targets and clinical diagnostic markers of osteosarcoma.Methods:1.According to the property that SP cells can efflux the fluorescent dye Hoechst 33342 and make the cells show weak fluorescence signal,SP and NSP cells in human osteosarcoma cell line MG-63 were isolated by flow cytometry.2.MTT assay and real-time quantitative polymerase chain reaction were used to verify the stemness of SP and NSP cells in MG-63 cells.3.The RNA,of SP and NSP cells were extracted and the differences and genes between them were detected by expression microarray.4.GO clustering and KEGG pathway enrichment were used to analyze the function of differentially expressed genes.5.Real-time fluorescence quantitative PCR was used to verify the expression of the top 10 differentially expressed genes.6.Construction of lentivirus plasmid with overexpression or silencing of candidate differentially expressed genes.7.The effects of candidate genes on the proliferation and differentiation of MG-63 cells were analyzed by growth curve and plate cloning experiment,and the effects of candidate genes on invasion and migration of MG-63 cells were analyzed by Transwell chamber and scratch assay.The relationship between candidate genes and the sensitivity of MG-63 cells to chemotherapeutic drugs was detected by MTT and apoptosis assay.Result:1.When the cell density was 9×105 cells/mL and the final concentration of Hoechst 33342 was 5 ?g/mL,the proportion of MG-63 SP cells was the highest(1.45% ± 0.23%).2.Compared with NSP cells,the expression of stem marker genes such as NANOG,SOX2 and Oct-4 in SP cells were up-regulated;the proliferation rate of SP cells was faster than that of NSP cells;the IC50 of SP cells was significantly higher than that of NSP cells,and the difference was statistically significant compared with NSP cells.Drug resistance related genes MRP,XRCC1,MGMT and ABCG2 were up-regulated in SP.3.The results of gene chip detection showed that a total of 7332 differential genes were detected in SP and NSP cells,of which 3661 genes were up-regulated and 3671 genes were down-regulated in SP cells.4.The results of GO cluster analysis showed that 55 terms were related to molecular function regulation.It mainly includes kinase activity(24%),channel activity(20%),transporter activity(20%),transmembrane transporter activity(18%),receptor activity(16%)and binding(15%).Differential genes are involved in 231 biological processes.It mainly includes functional regulation(33%),positive regulation(9%),negative regulation(8%),cell adhesion(3%),systematic process(3%)and cell biological process(6%).KEGG pathway enrichment results showed that differentially expressed genes were involved in 31 pathways.It is mainly PI3K-Akt signaling pathway,neuroactiveligand-receptor interaction,cytokine-cytokine receptor interaction,transcription disorders in cancer and so on.5.The results of PCR verification gene chip showed that among the ten differential genes selected and verified,the expression of two differential expressed genes was contrary to the microarray data,and the accuracy was 80%.6.Bioinformatics analysis combined with the results of real-time fluorescence quantitative PCR,KLK5,RAPH1 and TFPI2 were selected as candidate differentially expressed genes.7.MG-63 cells were transfected with lentivirus plasmid and the MG-63 cell lines with KLK5 overexpression(KLK5+)and negative control(KLK5-NC)were successfully constructed by puromycin screening.At the same time,the MG-63 cell line of RAPH1 silencing(shRAPH1)and its negative control(RAPH1-NC),and the MG-63 cell line of TFPI silencing(shTFPI2)and its negative control(TFPI2-NC)were also successfully constructed.8.The results of cell biological function test showed that compared with RAPH1-NC,the ability of cell proliferation,clone formation,migration and invasion of shRAPH1 was significantly increased,and the sensitivity to cisplatin was weakened.Compared with TFPI2-NC,the ability of cell proliferation,clone formation and migration of shTFPI2 was significantly increased,and the sensitivity to cisplatin was weakened.Compared with KLK5-NC,the ability of cell proliferation,clone formation,migration and invasion of KLK5+ was significantly increased,and the sensitivity to cisplatin was weakened.Conclusion:1.SP cells of human osteosarcoma cell line MG-63 sorted by flow cytometry have the characteristics of cancer stem cells,including self-renewal,the potential for differentiation and cisplatin resistance.2.KLK5 gene can promote the proliferation,differentiation,migration and invasion of osteosarcoma cells and reduce the sensitivity to cisplatin.The effect of RAPH1 and TFPI was opposite to that of KLK5 gene.3.KLK5,RAPH1 and TFPI2 may become targets and specific diagnostic molecular markers for the treatment of osteosarcoma.