| ObjectiveTo investigate the effects of the ethanol extract of Tibetan medicine Deziyan gxin(DZYX)on the proliferation and migration of gastric cancer cells,to further expl ore its molecular mechanism.MethodsGastric cancer cells were treated with DZYX ethanol extrac,and the contro l group(containing 1‰ DMSO)、DZYX ethanol extrac group(containing 200,400,600 μg/m L DZYX),and cisplatin group(10 μg/m L DDP)were set up.The number of g astric cancer AGS cells was observed by an inverted microscope under the effect of D ZYX.The effects of different concentrations of DZYX ethanol extrac on the proliferat ion of AGS cells were detected by cell counting kit-8 assay(CCK-8)、Ed U staining a nd real-time cellular analysis(RTCA).The effects of DZYX ethanol extrac on the AG S cell cycle were detected by propidium iodide(PI)single staining combined with flo w cytometry.RTCA cell migration assay,cell scratch assay,and Transwell assay were detected to the migration of gastric cancer cells.The change of cytoskeleton by confo cal microscopy.The change of Cyclin A,CDK2,p-AKT,p-mtor,and MMP-2 by west ern blot.Western blot to investigate p-m TOR,p-AKT,Cyclin A,CDK2,MMP-2 expres sion.Detection of differentially expressed genes in gastric cancer cells after treatment with DZYX ethanol extrac using transcriptome sequencing technology,and preliminar y investigated the possible mechanism of anti-gastric cancer effect through GO functio nal enrichment and KEGG signaling pathway enrichment analysis.Results1.Inverted microscope and CCK-8 results showed that different concentrations of DZYX ethanol extrac inhibited the proliferation of gastric cancer AGS and SCG-7901 cells in a concentration-dependent manner compared with the control group(P < 0.05).2.RTCA real-time cellular analysis showed that the proliferation curves of AGS cells were lower than the control group,and DZYX ethanol extrac could inhibit the prolifer ation of AGS cells(P<0.01).3.Ed U staining results showed that DZYX ethanol extrac inhibits the proliferation of AGS cells in a concentration-dependent manner(P < 0.05).4.Flow cytometry results showed that DZYX ethanol extrac blocks AGS cells in the S phase(P < 0.01)and down-regulated the expressions of Cyclin A and CDK2(P<0.05).5.RTCA cell migration assay showed that DZYX ethanol extrac inhibits the migration of SGC-7901 cells in a concentration-dependent manner(P < 0.05).6.The results of the scratch test showed that,compared with the control group,the DZ YX alcohol extract group had a larger scratch area and decreased wound healing rate a fter 24 h and 48 h of serum-free incubation;Transwell assay showed that the number of migrating gastric cancer cells was significantly reduced in the DZYX ethanol extrac t group,and DZYX alcohol extract significantly inhibited the migration ability of gast ric cancer cell line SGC-7901(P<0.05).7.The results of cytoskeleton staining showed that DZYX ethanol extrac can destroy t he polymerization of myosin,inhibit the formation of microfilaments,and reshape the cytoskeleton.8.Western blot results showed that DZYX ethanol extrac can significantly reduce the expression of PI3K/AKT/m TOR signaling pathway related proteins p-AKT and pm TOR(P < 0.05),the expression of matrix metalloproteinase 2(MMP-2)protein in gastric cancer cells was decreased(P < 0.05).9.Transcriptome sequencing screened differentially expressed genes in gastric cancer AGS cells after DZYX ethanol extract treatment,and a total of 2351 differentially exp ressed genes were screened,including 1470 down-regulated genes and 881 up-regulat ed genes.GO enrichment analysis in biological processes was mainly enriched in pro cesses such as DNA replication,G1/S cell cycle regulation,regulation of DNA replica tion initiation,etc.KEGG pathway enrichment analysis revealed that the pathways sig nificantly enriched in differential genes were cancer in pathway,PI3K-AKT signaling pathway,micro RNA,and RAS signaling pathway.Conclusions1.DZYX ethanol extract can inhibit the proliferation of AGS cells,induce S-phase blo ck and down-regulate the expression levels of S-phase associated phase protein Cyclin A,CDK2.2.DZYX ethanol extract significantly inhibits the migration ability of gastric cancer S GC-7901 cells3.The molecular mechanism by which DZYX ethanol extract inhibits gastric cancer c ells proliferation and migration may be related to the PI3K/AKT/m TOR pathway. |
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