The Role Of Raf, MEK, ERK On The Regulation Of NDRG1 Gene Expression And Invasion Process In Human Colorectal Carcinoma Cell Line In Vitro

Objective:To study and research the role of Ras/Raf/MEK/ERK pathway on the regulation of NDRG1 gene expression and invasion/migration process in human colorectal carcinoma cell lirie in vitro;analyzing the relationship among the ERK pathway,NDRG1 gene and tumor invasion/migration.Methods:Let human colorectal carcinoma cell line HT-29 co-culture with the Ras/Raf/MEK/ERK pathway correlated inhibitors.Then,use light microscope to observe cell morphology changes;use transmission electron microscope to observe the ultrastructure changes of the cells;use 24-transwell method to measure the invasion and migration(motion) abilities of the caner cells in vitro;the NDRG1 gene expression level was detected by immunocytochemical staining and western blot.Thus,we could explore the effects of the ERK pathway on the cellular morphology, invasion/migration abilities and the NDRG1 gene expression roughly.Results:After co-cultured with the inhibitors,the HT-29 colorectal cancer cells were less energetic and appeared with more dead cells;the cell outline was becoming regular in some degree,the size and appearance of the cells, nuclei were inclined with uniformity;the arrangement of the cells was also becoming regular which presented glandule-Iike texture with monolayer. There were more microvilli on the cell surface and the numbers of Golgi complexes,mitochondria were increasing;there were also more rough endoplasmic reticula;intracytoplasmic lumen was frequently seen;there were visible functional products(lipid droplet,microfilament bundle) in the cytoplasm;some cells presented cytoplasm vacuolization,nuclear chromatin aggregated on the verge of the nuclear membrane;showing a phenomenon that both had cell differentiation and apoptosis.Experiments in vitro showed,comparing with control groups,in each group co-cultured with inhibitor,the number of cells transferring through the polycarbonate membrane with 8μm pore in 24-transwell chambers decreased(P＜0.05),ever in chambers coated with Matrigel(measuring the ability of invasion) or without(measuring the ability of migration).The immunocytochemical staining results displayed that the NDRG1 gene expression level was low in HT-29 group and DMSO group,but in groups co-cultured with inhibitors there were significantly more NDRG1 proteins.The results of western blot showed NDRG1 protein band was seen in each group co-cultured with inhibitor except the control groups.Conclusions:1.Blocking the Raf/MEK/ERK pathway may induce differentiation and apoptosis of the HT-29 colorectal cancer cells.2.Blocking the Raf/MEK/ERK pathway may suppress the invasion and migration abilities of the HT-29 colorectal caner cells in vitro.3.Blocking the Raf/MEK/ERK pathway may up-regulate the expression of NDRG1 gene in the HT-29 colorectal caner cells significantly.