Mechanism Of The Regulation Of NDRG2 In Proliferation Of Colon Cancer Cells And Establishment Of NDRG2 Intestinal Epithelial Tissue-specific Knockout Mice

Human N-Myc downstream regulated gene2(N DRG2) is a tumor suppressor candidate identified by our laboratory. NDRG2 is highly expressed in brain、heart、liver、skeletal muscle and other tissues, and NDRG2 regulated cell proliferation 、 cell differentiation and cell apoptosis.NDRG2 had been investigated that it was suppressed in glioblastoma、breast cancer、hepatoma、gastric cancer、colon cancer and other malignant tumors, and it even could not be detected in many cancer lines. O verexpression of NDRG2 in MCF7 and Ca Co2 cells could suppress the cell proliferation. Other observation verified that NDRG2 could inhibit metastasis and proliferation of colon cancer, by regulating E-cadherin and TGF-β, and the association with relapse and 5-year survival was supported by our data. However, the functions of NDRG2 were not ful y discovered.Hepatocyte Growth Factor(HGF) is a multiple functional growth factor, which was involved in cell proliferation、cell differentiation、cell apoptosis、cell invasion and otherbiological progress via regulating PI3K/Akt、MEK/ERK、JAK/STAT signal pathway. Overexpression or mutational activation of c-MET was associated with malignant growth and metastasis in glioblastoma、breast cancer、colon cancer and many other tumors. Recent studies showed that HGF could promote the proliferation of alveolar type Ⅱ cells、melanoma cells、gastric cancer cells and other cell types, however, HGF could also inhibit the proliferation of some cancer cells like Hep G2、Huh7、HT29、HT115. The contrary functions of HGF on cell proliferation may be due to the crosslinking with other signal pathways.Transgenic mice had been invaluable for investigating the function of genes. The inhibition of cell proliferation by NDRG2 had been de monstrated, we established the Ndrg2 intestinal tissue-specific knockout mice via the Cre-Loxp system, and analyzed the influence of NDRG2 on the development of intestine and colon in mice. Therefore, we could possibly make it more clear of the functions of NDRG2 during the colon cancer initiation and development.Posttranslational modification is important for the functions of different proteins, particularly the protein acetylation was associated with degradation、transposition and secretion. We first analyzed the protein sequences of NDRG2 among different creatures, and we inferred three lysine sites to be the possible acetylation sites. And we established the point mutations to analyze the function of the acetylation on NDRG2.Our data demonstrated that NDRG2 suppressed the proliferation of colon cancer cells, therefore, we inferred that the inhibition was dependent on HGF/c-MET pathway. Besides, we found that the phenotype of the intestine and colon in Ndrg2 intestine tissue-specific knockout mice had changed obviously. The function of NDRG2 during the development of the intestine and colon in mice needed to be further explored.【Objectives】(1) To investigate whether the inhibition of cell proliferation by NDRG2 is t hrough regulating HGF/c-MET signal pathway.(2) To find out the variations of the phenotype in Ndrg2 intestine tissue-specific knockoutmice.(3) To demonstrate whether the posttranslational modifications affect the functions of NDRG2.【Methods】(1) Establish stable cell lines with overexpression of NDRG2, and analyze the affection on HGF/c-MET signal pathway through Real-time PCR and Western blot.(2) Establish the intestinal epithelial tissue-specific knockout mice, and find out the variations of the phenotype through Western blot and immunohistochemical staining.(3) Construct the expression vector of NDRG2 with point mutation and study the posttranslational modifications of NDRG2.【Results】(1) NDRG2 suppressed the proliferation of HT29 cells through regulating HGF/c-MET signal pathwayOverexpression of N DRG2 up-regulated HGF, thus facilitated the expression and phosphorylation of c-MET, which could stimulate the expression of p21 and p27. Cell proliferation rate would be inhibited.(2) NDRG2 affected the development of mice intestineThe expression of NDRG2 in intestinal epithelial tissue-specific knockout mice decreased obviously. And compared with wild type mice, the length of the intestine increased in intestinal epithelial tissue-specific knockout mice, possibly due to the increase of the villi.(3) The point mutation of NDRG2 expressed successfullyWe successfully established the expression vector of N DRG2 with K-Q(lysine-glutamine) or K-R(lysine-arginine) mutation. And we could investigate whether the function of NDRG2 was dependent on the acetylation on K171/K273/K288.【Conclusions】 NDRG2 inhibited the proliferation rate of the colon cancer cell line HT29 via regulating HGF/c-MET pathway; NDRG2 was involved in the intestinal development in C57/BL6 mice models, but the molecular mechanism needed to be further studied.Whether the function of NDRG2 depended on the acetylation would be further explored.