Study On The Role Of Differential Gene AKAP13/Rho Pathway In Osteosarcoma

Background: Osteosarcoma(OS)is a primary malignant bone tumor,commonly seen in adolescents,with a high degree of malignancy and poor prognosis.The tumor is locally invasive and can cause hematogenous lung metastasis,as well as metastasis to other organs and tissues.Early metastasis of osteosarcoma is an important factor causing high mortality in patients.Therefore,it is a key issue that needs to be addressed urgently to conduct more in-depth research on the mechanism of invasion and metastasis of osteosarcoma,find potential target genes and related proteins,and effectively inhibit the metastasis of osteosarcoma cells to other organs through targeted methods at an early stage.Bioinformatics is a newly emerging frontier discipline that has developed into the post genomic era.Its main research content includes large-scale data integration and visualization,genomics comparison,metabolic network analysis,data analysis of protein structure and function,gene expression profile network analysis,and drug target screening.This study uses bioinformatics analysis to screen potential target genes and related pathways for the treatment of osteosarcoma.Objective: This study uses bioinformatics technology to collect and analyze high-throughput gene chips from osteosarcoma patients present in the database,identify differential genes and related pathways between osteosarcoma and normal bone tissue,obtain potential target genes that regulate osteosarcoma,and preliminarily explore and study the role of this gene and related pathways in osteosarcoma,thus providing new research ideas and directions for the anti metastasis treatment of osteosarcoma in clinical practice.Methods: 1、Bioinformatics analysis(1)Search for gene chips from osteosarcoma patients from public databases such as GEO,TCGA,and DAVID,and appropriately screen them to obtain corresponding gene chip datasets.And appropriately group the selected gene chip dataset.(2)Conduct differential gene analysis on the grouped gene chip dataset to screen out the differential genes between osteosarcoma cells and normal cells,and select the genes with significant differences from them.(3)The selected genes were enriched and analyzed to find their related pathways.(4)Query the KEGG database for the most significant differential gene related pathways and their role in cells.(5)Conduct network analysis of differential genes in the String database,and mine upstream and downstream regulatory genes.(6)Query the expression amount of differential genes in sarcoma(SARC)in the TCGA database.(7)Propose reasonable assumptions about the regulatory role of the screened differential genes and pathways in osteosarcoma.2、Cell experiment(1)143B osteosarcoma cells were obtained and cultured and divided into three groups: experimental group(si AKAP13),negative control group(si NC),and blank control group(Blank).(2)Fluorescence microscopy is used to detect transfection effect.(3)CCK-8 method was used to detect the effect of AKAP13 knockdown on the proliferation ability of 143 B osteosarcoma cells.(4)The effect of AKAP13 knockdown on the invasion and migration ability of 143 B osteosarcoma cells was examined using a scratch test.(5)Western Blot experiment was used to detect the effect of AKAP13 knockdown on the expression of Rho A protein in 143 B osteosarcoma cells.Results: 1、Results of bioinformatics analysis(1)Finally,two gene chips,GSE125645 and GSE161407,were selected,and the gene data related to osteosarcoma patients were divided into three groups: A,B,and C.(2)Group A differential gene analysis results obtained 8734 gene probes with statistical significance.(3)Group B differential gene analysis results obtained 79368 gene probes with statistical significance.(4)Group C differential gene analysis results obtained 7140 gene probes with statistical significance.(5)The analysis results of common differential genes in groups A,B,and C showed that 17 common differential genes were obtained,among which the expression of PRDM10 gene was down regulated,and the expression of ZNF562 gene was significantly down regulated;FOXP4,HIVEP3,DGKZ,LOC100132215,G6PC3,JARID2,IFT140,TMEM204,PLEKHM2,LRRFIP1,SCNN1 A,NKX3-2,GIT2 gene expression was upregulated,while OTX1,AKAP13 genes were significantly upregulated.(6)KEGG database shows that AKAP13 acts on two pathways,called A-kinase anchor protein 13.As Rho-Guanine Nucleotide Exchange Factors(GEFs)and Rho-GTPase related proteins,AKAP13 plays an important role in the biological system,endocrine system,parathyroid hormone synthesis,secretion,and action,human diseases,viral infections,human cytomegalovirus infection,membrane transport,and other related aspects.(7)String database shows that AKAP13 can directly regulate downstream Rho A gene proteins.(8)TCGA database shows that AKAP13 gene expression in sarcoma(SARC)has significantly increased compared to normal tissue.2、Results of cell experiment(1)The fluorescence detection results showed that si RNA had been successfully transfected into 143 B osteosarcoma cells.(2)In the CCK-8 experiment,the proliferation ability of 143 B osteosarcoma cells in the experimental group(si AKAP13)was significantly decreased compared to the negative control group(si NC)and blank control group(Blank).(3)In the scratch experiment,the scratch healing rate of the experimental group (si AKAP13)was significantly lower than that of the negative control group(si NC)and blank control group(Blank).(4)WB results showed that the expression of Rho A protein in the experimental group(si AKAP13)decreased significantly compared to the negative control group(si NC)and blank control group(Blank)(P<0.05),indicating that AKAP13 knockdown can inhibit the expression of Rho A protein in 143 B osteosarcoma cells.Conclusion: The differential gene AKAP13,screened and analyzed from databases such as GEO and TCGA,is a potential target gene for regulating osteosarcoma cells.The AKAP13/Rho pathway may play a crucial regulatory role in the occurrence,development,migration,and invasion of osteosarcoma..When using si RNA to knock down AKAP13,it can inhibit the expression of Rho A protein in 143 B osteosarcoma cells,leading to certain inhibitory effects on the migration,invasion,and proliferation processes of 143 B osteosarcoma cells in vitro.This indicates that the AKAP13/Rho pathway plays an important regulatory role in the occurrence and metastasis of osteosarcoma.