| Objective To explore the role of enteric glial cells(EGCs)in the growth and metastasis of colon cancer.Methods1 In Vitro ExperimentCell experiments were performed in EGCs + CT26.WT group and CT26.WT group.A co-culture system of EGCs and CT26.WT cells was constructed using co-culture chambers or transwell chambers in the former group,while CT26.WT cells were cultured alone in the latter group.BrdU and MTT methods,the flow cytometry method,the invasion assay,and the scratch assay were employed to detect the effects of EGCs on CT26.WT cell proliferation,cell cycle and apoptosis,cell invasion,and cell migration,respectively.The expression of Caspase3 and Cleaved-Caspase3 in CT26.WT cells after EGCs culture supernatant intervention was examined by Western blot.2 In Vivo ExperimentsTwenty-eight SPF BALB/c inbred mice were randomly divided into four groups: the experimental group(EGCs group),the solvent control group(DMEM group),the model group(Model group),and the control group.The spleen of mice in the first three groups was injected with CT26.WT single cell suspension to construct CT26.WT cell transplanted tumor model,while the spleen of mice in the Control group was injected with the same amount of saline.Moreover,the EGCs group was injected intraperitoneally with 0.1mL EGCs culture supernatant,whereas the DMEM group was treated with 0.1 mL DMEM complete medium.Both the Model group and the Control group were treated with 0.1 mL sterile physiological saline.The weight of mice was measured every other day.After 2 weeks the mice were sacrificed,and the spleen,liver,brain,lung,and kidney were removed for visual and pathological examination of orthotopic tumorigenesis and metastasis.Immunohistochemistry was used to detect the expression of Ki67 in spleen situ tumor.The expression of DDR1,Vimentin,E-cadherin,HIF-2?/VEGFA,VEGFC and PTEN/PI3K-p110/AKT/MDM2/P53/P21 in spleen situ tumor and liver metastases tumor were detected by Western Blot.Results1 In Vitro Experiment1)Proliferation assay(BrdU,MTT): Both BrdU and MTT methods confirmed that compared with CT26.WT group,EGCs inhibited the proliferation of CT26.WT cells,and the difference was statistically significant(p <0.05);2)Invasion assay: Compared with CT26.WT group,EGCs inhibited the invasive ability of CT26.WT cells,and the difference was statistically significant(p <0.05);3)Scratch assay: Ccompared with CT26.WT group,EGCs inhibited the migratory ability of CT26.WT cells,and the difference was statistically significant(p <0.05);4)Cell cycle: Compared with CT26.WT group,CT26.WT cells G0 / G1 phase ratio in the EGC + CT26.WT group was increased,S phase was decreased,and G2 / M phase ratio was slightly increased,and the differences were statistically significant(p <0.05);5)Apoptosis: Compared with CT26.WT group,the early apoptosis rate of CT26.WT cells in the EGCs + CT26.WT group was increased,and the difference was statistically significant(p <0.05).There was no significant difference in the late apoptosis between the two groups;6)Western Blot: Compared with CT26.WT group,the expression of Caspase3 in CT26.WT cells in the EGCs + CT26.WT group was lower,while the expression of Cleaved-Caspase3 was higher,and the differences were statistically significant(p <0.05).2 In Vivo Experiments1)Weight: The mice body weight of EGCs group was significantly higher than that of DMEM group on the 12 th day and the 14 th day after operation,and the difference was statistically significant(p <0.05);2)Tumor formation and metastasis: all BALB / c mice in the EGCs group,the DMEM group,and the Model group showed visible in situ spleen tumor and liver metastases.DMEM group and Model group showed at least one pathological metastasis in renal,lung,or brain tissue,while EGCs group had no pathological changes of kidney,lung or brain tissue;3)Diameter of spleen in situ tumor: the spleen tumor diameter of EGCs group is less than that of DMEM group and Model group,and the difference was statistically significant(p <0.05);4)Ki-67 Immunohistochemistry of situ tumor in Spleen: the expression of Ki-67 in EGCs group was lower than that in DMEM group and Model group,and the difference was statistically significant(p <0.05);5)Western Blot analyses of spleen situ tumor: the expressions of E-cadherin,PTEN,P53 and P21 in the EGCs group were higher than those in the DMEM group and Model group,whereas expressions of DDR1,Vimentin and VEGFA were lower than those in the DMEM group.Moreover,the expressions of VEGFC,HIF-2?,PI3K-p110,AKT and MDM2 were lower than those in the DMEM and Model group,and the differences were statistically significant(p <0.05).6)Western Blot of Liver metastases tumor: the expressions of E-cadherin,PTEN,P53 and P21 in the EGCs group were higher than those in the DMEM group and Model group,while MDM2 expression was lower than in the DMEM group,and the expressions of DDR1,Vimentin,VEGFA,VEGFC,HIF-2?,PI3K-p110 and AKT were lower than those in the DMEM and Model group,and the differences were statistically significant(p <0.05).Conclusions1 EGCs can inhibit the growth and metastasis of CT26.WT colon cancer cells.2 EGCs inhibited the growth of CT26.WT colon cancer may be related to the role that EGCs play in promoting PTEN/PI3K/AKT/MDM2/P53/P21 signaling pathway and inhibiting HIF-2?/VEGFA signaling pathway.3 EGCs inhibited the metastasis of CT26.WT colon cancer may be related to the role that EGCs play in promoting the expression of E-cadherin,and inhibiting the expression of DDR1,Vimentin and VEGFC. |
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