Objective:Bioinformatics technology was used to screen anti-osteosarcoma cell drug small molecules,combined with in vitro cell experiment methods to explore whether the screened drug small molecules can inhibit the growth of osteosarcoma cells and promote cell apoptosis,and the mechanism of action was studied.Method:A study cohort containing osteosarcoma and its healthy controls was retrieved from GEO database,and GSE12865,GSE33383,GSE99671 and GSE126209 were used as chip sequencing data to screen out healthy control samples and osteosarcoma samples,and then their differences were analyzed.The results were cross-analyzed based on the jveen tool.In addition,GO and KEGG enrichment analysis was performed for GSE12865,GSE33383,GSE99671 and GSE126209 differential genes using cluster Profiler package.At the same time,GO and KEGG enrichment analysis was also performed for intersection genes.In addition,small molecular compounds with strong binding ability to target proteins were screened by computer simulation.Subsequently,based on the above predictive analysis results,we verified the HOS cell model by MTT assay,Trans Well assay,flow cytometry,RT-qPCR and western blotting.Result:33 interactional up-regulated genes and 106 interactional down-regulated genes were screened,and the GO of 33+2 interactional up-regulated differential genes and 106+4 down-regulated differential genes were analyzed.Using GSE12865 as chip sequencing data,1452 up-regulated and 1387 down-regulated differentially differentiated genes were screened.Using GSE33383 as chip sequencing data,499up-regulated and 506 down-regulated differentially differentiated genes were screened.Using GSE99671 as microarray sequencing data,274 up-regulated and 457down-regulated significantly differentiated genes were screened.Using GSE126209 as chip sequencing data,1158 up-regulated and 1321 down-regulated differentially differentiated genes were screened.The intersection of differential genes showed that the expression levels of 33 genes were significantly up-regulated in 3 study cohorts,and the expression levels of 2 genes were significantly up-regulated in 4 study cohorts at the same time.The expression levels of 106 genes were significantly down-regulated in three study cohorts simultaneously,and the expression levels of 4genes were significantly down-regulated in four study cohorts simultaneously.Then,the enrichment analysis of 33+2 overlapping up-regulated differential genes and106+4 down-regulated differential genes was performed.The results of MTT assay showed that with the increase of drug concentration and drug action time,the vitality of HOS cells decreased gradually,and the cell vitality was proportional to the drug concentration and drug action time.Trans Well experiments demonstrated that2,2,2,2,1-[(1S)-1-methyl-2,<>-ethylenediyl] dinitrile] tetramethylacetamide could reduce the invasion ability of HOS in osteosarcoma cells.Flow cytometry indicated that HOS death of osteosarcoma cells was related to cell cycle arrest.In RT-qPCR and western blotting,CDK4 m RNA expression was down-regulated and p21 and p18 m RNA expression was up-regulated in drug-treated osteosarcoma cells compared with blank group.The relative expression level of CDK4 protein in the dosing group was decreased and inversely proportional to the dosing concentration(**P < 0.01).The relative protein expressions of p21 and p18 increased,and were proportional to the dosage concentration(**P < 0.01).These results suggest that 2,2,2,2,1-[(1S)-1-methyl-2,<>-ethylenediyl] dinitrile] tetramethylacetamide can regulate cell cycle progression and promote apoptosis of HOS cells by inhibiting CDK inhibitors.The results of MTT assay showed that with the increase of drug concentration and drug action time,the vitality of HOS cells decreased gradually,and the cell vitality was proportional to the drug concentration and drug action time.Trans Well experiments demonstrated that 2,2,2,2,1-[(1S)-1-methyl-2,<>-ethylenediyl] dinitrile]tetramethylacetamide could reduce the invasion ability of HOS in osteosarcoma cells.Flow cytometry indicated that HOS death of osteosarcoma cells was related to cell cycle arrest.In RT-qPCR and western blotting,CDK4 m RNA expression was down-regulated and p21 and p18 m RNA expression was up-regulated in drug-treated osteosarcoma cells compared with blank group.The relative expression level of CDK4 protein in the dosing group was decreased and inversely proportional to the dosing concentration(**P < 0.01).The relative protein expressions of p21 and p18 increased,and were proportional to the dosage concentration(**P < 0.01).These results suggest that 2,2,2,2,1-[(1S)-1-methyl-2,<>-ethylenediyl] dinitrile]tetramethylacetamide can regulate cell cycle progression and promote apoptosis of HOS cells by inhibiting CDK inhibitors.Conclusion:The screened small drug molecules 2,2,2,1-[(1S)-1-methyl-2,<>-ethylenediyl] dinitrilyl] tetramethylacetamide can promote HOS apoptosis in osteosarcoma cells.It can up-regulate the expression of p21 and p18,thereby inhibiting CDK4 and triggering the inhibition of cell cycle progression. |