Ran Promotes Malignant Phenotype Of Colorectal Cancer And The Underlying Mechanisms

【Background】 Colorectal carcinoma is one of the most common malignant tumors in the world.Because of no symptoms or mild symptoms in early colorectal cancer patients,the majority of patients have occurred distant metastasis.Consequently,the five-year survival rate in patients with metastatic colorectal cancer is only 10%.Therefore,in order to achieve the individualized treatment,one or more tumor markers are needed to predict the prognosis and the therapeutic effect of the patients.the tumor to guide the patient in order to achieve the individualized treatment of the tumor.Ran,also named as Ras-related nuclear protein,is a protein that we found it can interact with Txl-2b in colon cancer cells.Ran plays an important role in the regulation of nucleocytoplasmic trafficking and mitosis in normal cells.Recent studies have shown that Ran is highly expressed in most tumors and can promote some malignant phenotypes of the tumors.However,the underlying mechanisms are still not well understood.【Objectives】1.To observe the expression of Ran in primary tumors,adjacent normal tissues and metastatic tumors of colorectal cancer and to observe the expression of Ran in colorectal cancer cells.2.To establish Ran down-regulated and overexpressing cell models.3.To observe the effect of Ran on the proliferation and apoptosis of colorectal cancer cells.4.To observe the effect of Ran on invasion and metastasis of colorectal cancer cells.5.To explore the molecular mechanisms of Ran promoting the malignant phenotype of colorectal cancer.【Methods】 1.To observe the expression of Ran in primary tumors,adjacent normal tissues and metastatic tumors of colorectal cancer and to observe the expression of Ran in colorectal cancer cells.1)Immunohistochemical staining technique is used to detect the expression of Ran in the primary tumors,adjacent normal tissues and metastatic tumors of colorectal cancer.The differences of Ran expression levels in the primary tumors,adjacent normal tissues and metastatic tumors were analyzed.2)Detect the expression of Ran in normal intestinal epithelial cells and various colon cancer cell lines and screen out the cell lines suitable for the establishment of cell models.2.To establish Ran down-regulated and overexpressing cell models1)Cells are transiently transfected with Ran-si RNA Oligos and then verify their interference efficiency.2)Ran-sh RNA lentivirus infects HCT116 and DLD-1 cell lines to establish Ran down-regulated cell models and then verify their interference efficiency.3)Ran-sg RNA lentivirus infects HT29 and SW480 cell lines to establish Ran overexpressing cell models and to verify their overexpression efficiency.3.To verify the effect of Ran on the proliferation and apoptosis of colorectal cancer cells1)Cell Counting Kit-8 is used to detect tumor cell proliferation in Ran down-regulated and overexpressing cell models.2)Flow cytometry is used to detect the apoptosis of tumor cells after Ran-si RNA Oligos transfection.3)Western blot is used to detect the expression of caspase-3 and PARP after Ran-si RNA Oligos transfection.4.To verify the effect of Ran on invasion and metastasis of colorectal cancer cells1)migration and invasion assay is used to detect tumor cell migration and invasion in down-regulated and overexpressing cell models.2)Nude mice tail vein transfer assay and small animal live imaging technique is used to detect tumor cell metastasis in vivo.5.To explore the molecular mechanisms of Ran promoting the malignant phenotype of colorectal cancer1)The expression of EGFR,p-EGFR,Akt,p-Akt,ERK and p-ERK in p53,β-catenin,NF-ΚB,EGFR and total protein in the nuclear protein of Ran interferon and overexpression are detected by Western blot.2)Real-time quantitative PCR is used to detect the expression of EGFR in Ran overexpressing cell models.3)Immunofluorescence is used to detect the expression of Ran and p-EGFR in DLD-1 cells after Ran-si RNA Oligos transfection.【Results】 1.The expression of Ran in the primary tumors of colorectal cancer are significantly higher than adjacent normal tissues(P=0.02).The expression of Ran in metastatic tumors are significantly higher than in primary tumors(P=0.0008).Ran expression in tumor cells is significantly higher than HIEC,both at m RNA level and total protein level.2.si RNA oligos are transfected into HCT116 and DLD-1 cell lines.The expression level of Ran protein in experimental group is significantly lower than negative control group.In HCT116 cells,si-1 and si-2 have the best down-regulated effect at 20 n M,and the interference efficiency is more than 70%.In DLD-1 cells,although the interference efficiency is not as good as HCT116,the interference efficiency is greater than 50%.The real-time quantitative PCR and Western blot showed that Ran in the experimental group are decreased by 71% and 59%,respectively.Ran’s loss-of-function models are established successfully.In SW480 and HT29,compared with the control group,the m RNA levels of Ran in the experimental group are increased by 3.5 times and 3.7 times.So Ran’s gain-of-function models are also established successfully.3.In the Ran down-regulation models,compared with the control group,the cell growth rate of HCT116 is significantly decreased from the 3rd day.From the 2nd day,the cell growth rate of DLD-1 Significantly reduced.In the overexpression models,the cell growth rate of the SW480 and HT29 is significantly higher than control groups.4.The early apoptotic rate of si-1 and si-2 is significantly higher than control groups(P<0.05).The late apoptotic rate of si-1 and si-1 is significantly higher than control groups.Compared with NC group,the expression of caspase-3 is not significantly changed,but the expression level of the cleaved caspase-3 is significantly increased.The expression of PARP is not significantly decreased,but the expression of cleaved PARP is significantly increased.5.Migration and Invasion assays show that in Ran down-regulated cell models,the migration and invasion ability of HCT116 and DLD-1 cells are significantly decreased compared with control group.In Ran overexpressing cell models,the migration and invasion ability of SW480 and HT29 cells are significantly increased compared with the control group.The results of tail vein transfer in nude mice show that in Ran down-regulated cell models,the metastasic ability of HCT116 cells is significantly decreased compared with the control group.The size of the lung metastases in the experimental group is significantly larger than that in the control group.6.Western blot shows that the expression of EGFR,β-catenin and NF-κB in the nucleus is significantly decreased and the expression of p53 in the nucleus is significantly increased.In Ran down-regulated cell models,The expression of p-EGFR,Akt,p-Akt,ERK and p-ERK are significantly decreased.In the Ranoverexpression models,The expression of EGFR,p-EGFR,p-Akt,ERK and p-ERK are significantly increased.Real-time quantitative RCR shows that the EGFR m RNA level of SW480 is up to 1.8 times higher than the control group,and the EGFR m RNA level of HT29-Ran is up to 1.9 times.Immunofluorescence shows that the fluorescence intensity of Ran is down by 72% and the fluorescence intensity of p-EGFR is reduced by 35% in DLD-1 cells.【Conclusions】 1.The expression of Ran in adjacent normal tissues,primary tumors and metastatic tumors was increased in turn,and Ran was highly expressed in colorectal cancer cell lines.2.Ran can promote the proliferation of colorectal cancer cells,and inhibit caspase-3-mediated apoptosis in vitro.3.Ran can promote the migration and invasion of colorectal cancer cells in vitro and in vivo.4.Ran may promote the malignant phenotype of the tumor by regulating the expression of EGFR and affecting the Ras/MAPK/ERK and PI3K/Akt signaling pathways.