| Gastric cancer has a high morbidity and mortality rate in the world.The occurrence and development of gastric cancer are regulated by genetic and environmental factors.The dysfunction of multiple signaling pathways contribute to the occurrence and progression of gastric cancer.CXCL8 is a multifunctional cytokine that is highly expressed in a variety of malignant tumor cells.CXCL8 promotes the proliferation?migration?invasion?angiogenesis and epithelial-mesenchymal transition of cancer cells.FAK is highly expressed in cancer and promotes the development of cancer by regulating cell migration and epithelial mesenchymal transformation.In this experiment,the gastric cancer cell lines MKN45 and FAK knockout gastric cancer cells(MKN45-FAK-/-)were used for transcriptome sequencing.The analysis of differentially expressed genes revealed that CXCL8 was significantly down-regulated in FAK knockout cells.Based on the essential roles of CXCL8 and FAK in the process of occurrence and development of gastric cancer,we explored?1?whether the expression of CXCL8 was regulated by FAK in gastric cancer cells,?2?the promotion effect of CXCL8 on cell proliferation,colony formation,cell migration and EMT of gastric cancer cells,?3?the roles of FAK pathway in the process of cell proliferation,colony formation,cell migration and EMT stimulated by CXCL8.Part one:The sequencing analysis and verification of transcriptome,?1?Transcriptome sequencing analysis of MKN45 and MKN45-FAK-/-cell lines,from the dataset,277 DEGs between MKN45 and MKN45-FAK-/-were identified,the functional annotation and potential pathways of differentially expressed genes?DEGs?were additionally examined by Gene Ontology?GO?and Kyoto Encyclopedia of Genes and Genomes?KEGG?enrichment analyses.From the list of differentially expressed genes,the expression of CXCL8?**p<0.01?was down-regulated;?2?The technical validation of CXCL8 expression was performed by qPCR and ELISA,and results showed that the expression of CXCL8 was significantly down-regulated in MKN45-FAK-/-cells?**p<0.01?;?3?The expression of CXCL8 was decreased?**p<0.01,*p<0.05?in gastric cancer cells detected by qPCR and Western blot after treated with TAE226;?4?The expression of CXCL8 detected by qPCR and ELISA was significantly increased?**p<0.01,*p<0.05?after overexpressing of FAK.Part two:The promotion effect of CXCL8 on cell proliferation,colony formation,migration and EMT of gastric cancer cells.?1?The expression of receptor CXCR1/2 of three gastric cancer cells were detected by Western blot.The receptor CXCR1 was detected in all three gastric cancer cells,and CXCR2 was almost undetectable;?2?the effect of CXCL8 on proliferation,colony formation and migration were detected by MTT,trypan blue exclusion assay,clone formation assay,transwell and scratch assay.The CXCL8 promote the proliferation,colony formation and migration of gastric cancer cells?**p<0.01,*p<0.05?;?3?The expression of EMT related proteins were detected by Western blot,and EMT of gastric cancer cells was promoted by treatment of CXCL8,the expression of E-cadherin was down-regulated?**p<0.01,*p<0.05?and the expression of N-cadherin was up-regulated?*p<0.05?.Part three:The roles of FAK pathway in the process of cell proliferation,colony formation,cell migration and EMT stimulated by CXCL8.?1?Activation of the FAK pathway by CXCL8 was detected by Western blot,and the phosphorylation level of FAK was increased by CXCL8 treatment?*p<0.05?;?2?After FAK suprressed by TAE226,the effect of CXCL8 on proliferation,clone formation and migration were detected by MTT,trypan blue exclusion assay,clone formation assay,transwell and scratch assay.The results showed that the promotion effect of CXCL8 on the cell proliferation and migration were significantly?**p<0.01?suprressed by FAK inhibitior TAE226 in gastric cancer cells;?3?The EMT induced by CXCL8 in MKN45 and MKN45-FAK-/-was detected by Western blot and the effect of CXCL8-induced MKN45-FAK-/-EMT was suppressed compared with MKN45.Summary,the expression of CXCL8 was regulated by FAK pathway in gastric cancer cells;The CXCL8 receptor CXCR1 were detected in three kinds of gastric cancer cells;The proliferation,migration,colony formation and epithelial mesenchymal transformation of gastric cancer cells were promoted by CXCL8;FAK can be activated by exogenous addition of CXCL8,which indicated that CXCL8 has a certain regulatory effect on MKN45;The promotion effect of CXCL8 on proliferation,migration and EMT of gastric cancer cells were weakened by FAK inhibition.Therefore we propose that the cell proliferation,migration,colony formation and EMT of gastric cancer cells might be regulated by CXCL8/FAK signal pathway regulating.This study will provide theoretical basis and technical support for elucidating the pathogenesis,diagnosis and treatment of gastric cancer. |
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