Molecular Mechanism Of Epithelial-mesenchymal Transition Elicited By GNL3 In Colon Cancer Cells

BACKGROUND AND OBJECTIVES: Colon cancer is one of the most common malignant tumors in the world,of which the morbidity and mortality are located in the forefront.Treatment failure and death from cancer is mainly caused by metastasis of cancer.In the process of tumor metastasis,invasion and migration of tumor cells are the critical steps.According to recent studies,epithelial-mesenchymal transition(EMT)plays an important role in the process of tumor metastasis,thus becoming the research hotspot in the field of tumor metastasis.EMT refers to a process of transformation of epithelial cell to mesenchymal cell phenotype,and it is closely related to the development of embryos,tissue repair,tumor metastasis,maintenance of stemness,inhibition of cell apoptosis and senescence,and immunosuppression.The main features of EMT are deformation of epithelial cell,loss of polarity,decreased intercellular tight junction and adhesion ability.Epithelial cell is endowed with migratory and invasive properties,which translates into cell with the phenotype and properties of mesenchymal cell.Study on the molecular mechanism of EMT in colon cancer cell,we can not only expand the understanding of the biological behaviors of colon cancer metastasis,and may provide potential therapeutic strategies for the treatment of colon cancer.G protein nucleolar 3(GNL3),a nucleolar GTP-binding protein,is highly expressed in progenitor cells,stem cells,and in various types of cancer cells,while is a low expressed or not expressed in normal tissues and cells.Previous studies reported that GNL3 played a crucial role in cell proliferation,cell cycle regulation,inhibition of differentiation,biogenesis of ribosome,and maintenance of stemness,genome stability and telomere integrity,et al.Moreover,recent studies have shown that GNL3 is involved in the invasion and metastasis of cancers.However,the role of GNL3 in the invasion and metastasis of colon cancer remains unclear.Therefore,this study was performed to clarify it.Firstly,we tested the expression levels of GNL3 and EMT-related markers(E-cadherin,N-cadherin and vimentin)in colon cancer tissue specimens,analyzed the relationship between the clinical pathological parameters of patients with colon cancer and the expression of GNL3,and demonstrated the significance of GNL3 expression in the prognosis of patients with colon cancer.Secondly,we used interference vectors of GNL3 to transduce the colon cancer cells with high expression levels of GNL3,and established colon cancer cell lines with stable low expression levels of GNL3.Meanwhile,we used overexpression vectors of GNL3 to transduce the colon cancer cells with low expression levels of GNL3,and established colon cancer cell lines with stable high expression levels of GNL3.Then,we detected the changes of molecular and cellular biology in colon cancer cells with or without GNL3 knockdown or overexpression.Finally,we explored the possible molecular mechanism of EMT elicited by GNL3 in colon cancer cells.METHODS:(1)Immunohistochemistry and western blot were performed to examine the expression levels of GNL3 in colon cancer tissues and corresponding normal tumor-adjacent tissues.Moreover,the relationship between the clinical pathological features of patients with colon cancer and the expression of GNL3 was analyzed.(2)Western blot assays were performed to examine the expression levels of GNL3 in colon cancer cell lines.After cells were transduced with interference vectors or overexpression vectors,MTT assays,clone formation assays,cell invasive assays,scratch test,western blot and xenograft model in nude mice were conducted to detect the effects of the changes of GNL3 expression on cell proliferation,clone formation,invasion,migration and EMT in colon cancer cells,and the growth of tumor in vivo.(3)Western blot,immunofluorescence staining and Li Cl were used to explore the molecular mechanism of EMT elicited by GNL3 in colon cancer cells.RESULTS:(1)The expression levels of GNL3,N-cadherin and vimentin in colon cancer tissues were significantly higher than those in the corresponding normal tumor-adjacent tissues;E-cadherin exhibited an opposite trend.The expression of GNL3 was closely associated with differentiation,serosa invasion,lymph node metastasis and vascular invasion of colon cancer.The Kaplan-Meier survival analysis showed that the expression levels of GNL3 were markedly related with the prognosis of patients with colon cancer.(2)By transfection of exogenous vectors to down-regulate or up-regulate the expression of GNL3,we found that overexpression of GNL3 promoted the cell proliferation,invasion,migration and EMT in colon cancer cells.However,the knockdown of GNL3 had an opposite trend.(3)Overexpression of GNL3 made the ?-catenin enter the nucleus and activated Wnt/?-catenin signaling pathway,and induced EMT.Knockdown of GNL3 inhibited the EMT and Wnt/?-catenin signaling pathway,but this situation could be partially reversed by LiCl.CONCLUSION: The expression levels of GNL3 are higher in colon cancer tissues compared with that in corresponding normal tumor-adjacent tissues,and the expression of GNL3 is associated with the invasion and metastasis of colon cancer.GNL3 is an important contributor to EMT in colon cancer cells.Overexpression of GNL3 promotes EMT via activation of Wnt/?-catenin signaling pathway,and contributes to invasion and migration in colon cancer cells.