Regulation Of VHL On Invasion And Metastasis Of Colorectal Cancer Stem Cells And The Effect Of Yiaifang

Object:1.The microsphere model of colorectal cancer(CRC)HCT116 and HT29 stem cells(CSCs)was constructed and identified by CRC CSC markers CD133,CD44,and Lgr5.The expression of CD133,CD44,and Lgr5 markers in CRC was investigated;the invasion and metastasis abilities of HCT116 and HT29 CSCs,and the ability of subcutaneous tumor formation in nude mice were determined;and the effect of the Yiaifang on the invasion and metastasis of HCT116 and HT29 CSCs was also investigated.2.The stable transfection HCT116 of high VHL expression was established,the role of VHL expression on the abilities of microsphere formation,invasion,metastasis,and metastatic tumor formation of HCT116 CSCs,the impact of VHL expression on HCT116 CSCs markers CD133,CD44,and Lgr5,and whether the Yiaifang can act on VHL and enhance its effect were determined.Methods: 1.Two CRC cell lines HCT116 and HT29 were used by serum-free microspheres to construct CSC model in CRC and induced its differentiation under serum-containing conditions.2.The CSCs markers CD133,CD44 and Lgr5 were identified by flow cytometry and Western blot,using single-layer adherent cells as the control group,and the expression of CSCs markers was detected.3.Using single-layer adherent cells as the control group,Transwell method was used to observe the invasion and metastasis abilities of CRC CSCs,and the ability of subcutaneous tumor formation in nude mice.4.Role of the Yiaifang on the abilities of microsphere formation,invasion and metastasis of CRC CSCs,and the expression of CSCs CD133,CD44 and Lgr5 were observed and in nude mice as well as for the impact of invasion and metastasis formation by tail vein injection.5.The stably transfected high VHL expression in CRC HCT116 was constructed bylentivirus infection.CRC HCT116 was infected with VHL empty lentivirus as a control group.The effect of high VHL expression on the microsphere-forming ability of CRC HCT116 stem cells was observed.6.CRC HCT116 was infected with VHL empty lentivirus as a control group.Transwell method was used to explore the invasion and metastasis ability of the stably transfected CSCs of VHL high expression in CRC HCT116,and in nude mice as well as for the invasive potential and metastatic capacity via tail vein injection;7.Using flow cytometry and Western blot to identify CD133,CD44 and Lgr5 markers in the stably transfected VHL high expression in CRC HCT116,CRC HCT116 was infected with VHL empty lentivirus as a control group to detect the expression of CSCs markers.8.The Yiaifang on the abilities of microsphere formation,invasion and metastasis of CSCs in the stably transfected strain of VHL high expression in CRC HCT116 and the expression of CD133,CD44,and Lgr5 markers were investigated,as well as in nude mice for the impact of the invasive potential and metastatic capacity by tail vein injection.Results:1.The number of invasion and metastasis of CRC stem cells HCT 116 and HT29 was more than that of single-layer adherent cells.CRC stem cells showed higher expression of CD133,CD44 and Lgr5 markers than single-layer adherent cells;2.Yiaifang can inhibit the number and size of microspheres of HCT116 and HT29colorectal cancer CSCs,the number of invasion and metastasis cells,the expression of CSCs markers CD133,CD44,Lgr5,and the number of invasion and metastasis of CSCs injected into the tail veins of nude mice;3.High VHL expression inhibited the number and size of microspheres of HCT116 and HT29 colorectal cancer CSCs,the number of invasion and metastasis cells,the expression of CSCs markers CD133,CD44,Lgr5,and the number of invasion and metastasis of CSCs injected into the tail veins of nude mice;4.Yiaifang can promote the expression of VHL.Conclusions:1.CRC CSCs exhibited stronger microsphere-forming ability,invasion and metastasis ability and the expression of stem cell markers were more abundant than single-layer adherent cells;2.VHL can suppress the stemness of CRC CSCs,thereby inhibiting microsphereforming ability,invasion and metastasis ability,and the expression of stem cell markers in colorectal cancer CSCs,and further inhibited the invasion and metastasis of CRC CSCs;3.Yiaifang can inhibit the stemness of CRC CSCs,thereby suppressing CRC CSCs invasion and metastasis formation.Yiaifang could promote VHL expression and enhance its effect.