| Rsearch pnrpose To study the effect of cantharidin,an effective ingredient of Mongolian medicine alaga-banbu,on the proliferation,invasion,migration and other biological behaviors of gastric cancer AGS cells,and to further explore the possible molecular mechanism of the effect of cantharidin on gastric cancer AGS cells by analyzing the differential mRNA group after the act of cantharidin on AGS cells.The research methods The effective coomponent of alaga-banbu was extracted by hot reflux method,quenching method and mixed extraction method.After the purity analysis by HPLC,the extracted cantharidin was applied to gastric cancer AGS cells,and the inhibition of gastric cancer AGS cell proliferation was detected by cck-8 method,and the IC50 value of 48h was determined.After the concentration of IC50 was applied to gastric cancer AGS cells for 48h,the effect of cantharidin extract on the migration ability of AGS cells in gastric cancer was detected by scratch healing experiment;Transwell chamber method was to detect the effect of cantharidin extract on the invasion ability of gastric cancer AGS cells;The effect of cantharidin extract on apoptosis of gastric cancer AGS cells was detected by fluorscence microscopy and Annexin V-APC&PI double staining;PI-FAC S was used to detect the effect of cantharidin extract on gastric cancer AGS cells’ cell cycle.High-throughput sequencing technique was used to detect the transcriptome changes of gastric cancer AGS cells before and after the action of cantharidin,and differential mRNA expression was detected.GO functional enrichment analysis and KEGG pathway enrichment analysis were conducted to analyze the molecular functions,signaling pathways and metabolic pathways that diffrentially expressed mRNA might participate in the anti-gastric cancer process.The results of the studg By comparison,the mixed extraction method has been the best.extraction method on cantharidin,with the highest purity of 87.8%.Compared with the Blank group,the results of CCK-8 method showed Cantharidin,an effective extract of Mongolian medicine alaga-banbu,can inhibit the proliferation of gastric cancer AGS cells in a time-dependent and dose-dependent manner.The IC50 value of cantharidin at 6.948μmol/L.After the action of 6.948 μmol/L cantharidin on gastric cancer AGS cells for 4811,apoptotic cells were detected under fluorescence microscope,Annexin V-APC&PI and PI-FACS detection showed that cantharidin can induce early and late apoptosis of AGS cells in gastric cancer and block G2/M stage.Compared with the Blank group,the apoptosis rate increased from 2.47 to 5.63(P<0.05),and the percentage of G2/M cells increased from 10.01 to 15.25(P<0.01).As detected by high-throughput sequencing technology,a total of 9,623 differentially expressed mRNA were found,of which 4,669 were up-regulated and 4,954 were down-regulated.Through literature review and enrichment analysis,19 mRNA that may be the key response genes of gastric cancer AGS cells acting on cantharidin were selected,among which 8 were up-regulated and 11 were down-regulated.The research couclusion The method of mixed extraction is an effective method to extract cantharidin from alaga-banbu.At 6.948 μmol/L,cantharidin inhibited the proliferation,migriation and invasion of AGS in gastric cancer cells.This may be related to cantharidin induced cell cycle arrest and apoptosis in gastric cancer.The mRNA expression patterns of gastric cancer cells were changed after the action of cantharidin,and these differentially expressed genes could affect the processes of gastric cancer proliferation and invasion though a vari ety of biological pathways.Through screening and combining with literatures,it was found that cantharidin may play an inhibitory role on biological behaviors such as proliferation,migration,invasion and cell cycle relation of gastric cancer by up-regulating MAP2K,CDKN1A,RASD1,DDIT4 and down-regulating ZNF121,STAT1 and CDK1. |
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