Activated Mutant SHP-2 Tyrosine Phosphatase Enhances Intestinal Tumor LOVO Cells Malignant Biological Behavior Via EMT

[Backgrounds]Cancer is a major disease as a threat to human health.In recent years,trends of increased incidence and early age of cancer have been discovered.Mounting evidences suggested that the development process from healthy cells to malignant cells,is under the influence of many factors and multi-step development during a long time,and is a gradual process with the participation of a variety of genes.Therefore,to study the role of genetic factors in the development of its tumorigenesis is of particular importance.Colon cancer is the most common gastrointestinal cancer in developed countries,and is also one of the common malignant tumors in China.In the past few years,the incidence and mortality of colon cancer are rising in many countries and regions.In our urban areas,incidence and mortality of this disease are the forefront.Nevertheless,the definite mechanisms of pathology and molecule biology of colon cancer is not entirely clear.SHP-2(Src homology 2-containing protein tyrosine phosphatase)is a non-receptor tyrosine phosphatases which encoded by the gene PTPN11.SHP-2 is involved the physiological processes in cell survival and migration.SHP-2 mutant has been reported as a crucial role in Noonan syndrome and various types of leukemia.At the same time,SHP-2 mutants have also been found in multiple solid tumors.D61G activation is one of the common activating mutations of SHP-2.D61G activation can lead to a solution of the binding site of N-SH2 and PTP domain,and cause increased activity of SHP2 tyrosine phosphatase,thereby contributing to the activation of downstream signaling pathways.Epithelial-mesenchymal transitions(EMT)means that,polarized epithelial cells lose their polarity in the case of specific pathophysiological,thereby to be transformed into mesenchymal cells,and to get activity and enhanced migration.The occurrence of EMT is induced by a complicated procedure with multiple genes,multiple steps and multiple stages.Meanwhile,EMT was thought to be the key event of invasion and metastasis in tumor cells,including colorectal carcinoma.But whether mutant SHP2-D61G can affect EMT and then promote the proliferation and metastasis of colon cancer cells have not yet been reported clear.Based on the previous research,the present study was designed to investigate the role of SHP2-D61G mutation on proliferation and metastasis of colon cancer by EMT.[Methods](1)SHP-2 + and SHP-2D61G plasmids were transfected into human colon cancer LOVO cells to construct stable transfected cell model.(2)MTT assay and plate clonality assay were performed to determine the number of viable transfected cells.The ability of anchorage-independent growth of transfected cells was determined by soft agar colony formation assay.And adhesion test,wound healing assay and transwell assay were employed to investigate the adherence activity and migration of transfected cells.(3)The expressions of EMT associated proteins were deternined before and after transfection and their correlation with EMT were analyzed.[Results](1)Transfected LOVO cell model of SHP-2 + and SHP-2D61G were successfully constructed.(2)Effect of SHP-2D61G mutation on the growth of colon cancer cells:MTT results showed that,cell proliferation rate increased significantly after transfection of SHP-2D61G mutant.And results of plate clonality assay and soft agar colony formation assay also showed that cell colony formation rate was more than the control group after transfection of SHP-2D61G mutant.Results above suggested that SHP-2D61G mutations could promote the clone formation and proliferative capacity of cancer cells.(3)Effect of SHP-2D61G mutation on the invasion and metastasis of colon cancer cells:Wound healing assay showed that,migration rate of cells transfected with SHP-2D61G mutant was significantly higher than that of the control group.Results of adhesion and transwell assay showed that the adhesive capacity and invasion rate of transfected cells were visibly higher when compared to the control groups.So SHP-2D61G mutation may have the ability to enhance cell migration and of colon cancer cells in vitro.(4)SHP-2D61G mutation leads to the occurrence of EMT:EMT-related markers which were detected by Western blot showed that EMT epithelial markers of SHP-2D61G mutation group were downregulated while the mesenchymal-like markers were increased.[Conclusion](1)SHP-2D61G could promote the proliferation,invasion and migration of LOVO cells.(2)EMT may play an important role in the process of SHP-2D61G affecting the biological behavior in human colon cancer LOVO cells.