| Objectives: This experiment identified the key genes in the occurrence and development of skin melanoma through bioinformatics analysis,and further explored the effect and mechanism of action of the key gene(TUBB4A)on the proliferation and migration of melanoma cells.Moreover,it analyzed the effects of two targeted drugs(Dihydroartemisinin and Nocodazole)of TUBB4A on melanoma cells.This study may provide new ideas for the pathogenesis and treatment of skin melanoma.Methods: We searched skin melanoma gene microarray data sets GSE46517,GSE15605 and GSE3189 of GEO database,and their transcriptome data was downloaded.a set of differentially expressed genes were screened out with R.Gene Ontology,KEGG pathway enrichment analysis and survival analysis were performed to obtain the significant function annotations and pathways of key genes.We downloaded the gene expression data of melanoma patients from the TCGA database to analyze the prognosis of the key gene TUBB4A in skin melanoma patients.Select two kinds of melanoma cell lines: A375 and B16-F10,transient transfection technique was used to down-regulate the expression of endogenous TUBB4A gene and screen melanoma cell lines transfected with TUBB4A gene. By real-time fluorescent quantitative PCR,CCK8,scratch test and Transwell migration experiment to detect the expression of TUBB4A in melanoma cells before and after transfection of si RNA,and the changes in the biological characteristics of melanoma cell lines,such as proliferation and invasion.Predict the targeted drugs of TUBB4A through Gene Cards database and conduct cell drug delivery experiments.Flow cytometry detects cell apoptosis and cell cycle.Results: We screened a total of 165 differential genes up-regulated and 393 differential genes down-regulated between skin melanoma and healthy controls.Differentially expressed genes are mainly involved in epidermal and skin development,keratinocyte and epidermal cell differentiation and other biological functions,as well as cancer transcription disorders and p53 signaling Pathways,prostate cancer and melanogenesis.After survival analysis,seven key genes,TUBB4A,HJURP,PSEN2,QPRT,SLC45A2,TRPV2 and UBE2 S,were screened out.Among them,the gene of TUBB4A has the most significant correlation with the prognosis of patients(P = 0.000039),and the prognosis of patients with high expression of TUBB4A was significantly lower than that of low expression patients.After transfecting melanoma cell lines A375 and B16-F10 with siRNA,the expression level of TUBB4A was significantly lower than that of the control group.Conduct cell function experiments to explore the effect of TUBB4A on the proliferation and migration of melanoma cells: CCK8 experiment results showed that the cell activity and proliferation of the si-TUBB4A group were significantly inhibited(P <0.01);the results of the scratch experiment showed that compared with the control group The cell migration distance of the si-TUBB4A group was significantly reduced(P <0.01);the results of the Transwell experiment showed that the number of cells passing through the membrane in the si-TUBB4A group was significantly lower than that of the control group,and the difference was statistically significant(P <0.01).After treating the A375 cell line with the targeted drugs dihydroartemisinin and nocodazole of TUBB4A,the morphology of tumor cells changed significantly,and the proportion of apoptosis was significantly higher than that of the control group(P <0.05).G2/ M The proportion of tumor cells in?stage is significantly increased.Conclusions: Highly expressed TUBB4A is associated with poor prognosis of melanoma patients,identified by bioinformatics analysis.TUBB4A promotes the proliferation and migration of A375 and B16-F10 melanoma cells.Its targeted drugs Dihydroartemisinin and Nonocodazole promoted the apoptosis of melanoma cells and made the tumor cells significantly blocked in G2/M stage.TUBB4A may be used as a potential clinical diagnosis and treatment target and prognostic marker for melanoma. |
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