Identification Of The Molecular Mechanisms And Target Proteins Of Nrip2in Colorectal Cancer Cells

Colorectal cancer is one of common malignant tumors which ranks as the third most common tumor-related cause of death in western countries and the incidence of colorectal cancer has recently risen in China. Studies have shown that colorectal cancer stem cells existed in colorectal cancer may be the cause of colorectal cancer occurrence and progression. We previously found that a novel Nrip2appeared knocking down in CD133+colorectal cancer cells and overexpression of Nrip2significantly inhibited the tumorigenicity of CD133+colorectal cancer cells in NOD/SCID mice. It suggested that knockdown of Nrip2in colorectal cancer stem cells may be a new molecular mechanism to promote the tumorigenicity and Nrip2could be a new potential target for intervention of colorectal cancer stem cells. However, the molecular mechanism of Nrip2in colorectal stem cancer cells has not yet been elucidated.ObjectiveIn this study, we explore the molecular mechanisms and the regulation of pathways of Nrip2and identify targeting proteins interacted with Nrip2basing on our previous achievements. This study will provide a foundation for developing targeted drugs with low toxicity for colorectal cancer.Method1.Nrip2-pEGFP-C1plasmid was constructed and transfected into293T cells. The cellular localization of Nrip2was observed by confocal microscopy.2.To study the molecular characteristics of Nrip2, phosphorylation sites of Nrip2were predicted by bioinformatics and confirmed by western blot. After the amino acids were changed by site-directed mutation, the cellular localization of mutants was observed by confocal microscopy.3.To study the relationship between Nrip2and Wnt pathway, we detected β-catenin activities of Nrip2and mutants by dual-luciferase reporter assay system.4.Nrip2was overexpressed in SW620to observe the tumorigenicity in mice.5.To identify the target proteins of Nrip2, interacting proteins with Nrip2were pulled down by immunoprecipitation. The proteins were identified by western blot.Result1.Nrip2distributed in the cytoplasm, nucleus, and lysosomes.2.S34and T152of Nrip2respectively could be phosphorylated by Akt and PKA kinase, suggesting that Nrip2could be regulated by Akt and PKA kinase. Changing the amino acid of S34to A34and T152to A152had no significant effect on cellular localization.3.Wild-type of Nrip2and mutants of S34A could inhibite p-catenin activity and mutants of T152A had no significant effect on β-catenin activity.4.Overexpression of wild-type Nrip2inhibited the tumorigenicity.5.We identified that HSP73, PKM2and FZD1were interacted with Nrip2. Conclusion:1.Wild-type of Nrip2and the mutants of S34A and T152A distribute in the cytoplasm, nucleus, and lysosomes.2.S34and T152of Nrip2respectively could be phosphorylated by Akt and PKA kinase3.Overexpression of Nrip2can inhibit the tumorigenicity.4.HSP73, PKM2and FZD1are interacted with Nrip2.5.Nrip2may participate in the progression of colorectal cancer by modulating Wnt pathway.