Study On Mechanism Of Lurap1 To Inhibit The Malignant Phenotype Of Non-small Cell Lung Cancer

Lung cancer is one of the main causes of death in malignant tumors.Although the treatment of lung cancer has made great progress in recent years,the long-term survival rate of patients is still not high due to the complex factors such as epigenetics,which play an important role in tumor growth and invasion.Therefore,it is an important task to discover new oncogenes/tumor suppressor genes and elucidate their mechanism of action.In recent years,the relationship between Wnt and Hippo signaling pathway and the occurrence and progression of tumor has been paid more and more attention.Wnt signal is a highly conserved signal transduction pathway in the process of species evolution,which is closely related to the physiological processes of embryo development,cell proliferation,differentiation and apoptosis.In addition,hippo signaling pathway is the first signaling pathway found in Drosophila,which is highly conserved in the evolution of species and closely related to the regulation of organ volume,tissue regeneration,cell polarity and tumor formation.There are also extensive interactions between the Hippo signaling pathway and Wnt signaling pathway,in which Dvl is one of the key node proteins.The latest research shows that in zebrafish,lurap1 can interact with Dvl2,and the combination of the two can inhibit the membrane recruitment of Dvl,and then affect the formation of zebrafish cytoskeleton.So in lung cancer,can lurap1 interact with Dvl to regulate Wnt and Hippo signaling pathway activity,and then affect the malignant phenotype of lung cancer cells?This aroused our great interest.Objective:To investigate the expression of Lurap1(leucine rich adaptor protein1)in NSCLC(non-small cell lung cancer),and its effect on proliferation and invasion,as well as its mechanism.Methods:1.Immunohistochemistry and immunofluorescence were used to analyze the expression of Lurap1 in lung cancer and its relationship with clinicopathological factors.2.MTT,colony formation,Transwell and other experimental methods were used to observe the effect of two-way regulation of lurap1 expression on the biological function of proliferation and invasion of lung cancer cells.3.Western blot was used to observe the effect of two-way regulation of lurap1 expression on Wnt and Hippo signaling pathwayand its possible mechanism.Results:1.The expression of lurap1 was positive in bronchial epithelial cells and normal bronchial epithelial cells(86.5%,32/37),but decreased in lung cancer tissues and cell lines(51.02%,75 / 147).Statistical results showed that the high expression of lurap1 was positively correlated with the differentiation of lung cancer,and negatively correlated with TNM stage and lymph node metastasis.The survival analysis provided by TCGA database showed that the survival time of patients with high expression of lurap1 was significantly longer than that of patients with low expression.2.In A549 and H1299 cell lines,overexpression of lurap1 inhibited cell proliferation and invasion(P<0.05),while siRNA-mediated Lurap1 knockdown promoted cell proliferation and invasion(P<0.05).3.Lurap1 can inhibit the activity of Wnt signaling pathway.We found that the expression of p-dvl2,p-gsk3?,cyclin D1 and c-myc were down regulated,while?-Catenin was down regulated when Lurap1 was overexpressed in A549 and H1299 cell lines.SiRNA-mediated Lurap1 knockdown in A549 and H1299 cell lines Showed the opposite results.4.Lurap1 can activate the activity of Hippo signaling pathway.We found that the expression of key phosphorylation cascade protein p-mst1,p-lats1 and p-yap in the Hippo signaling pathway was up-regulated,the expression of downstream target gene cyclin E and CTGF was down regulated,while the Yap in the nucleus was decreased when Lurap1 was overexpressed in A549 and H1299 cell lines.SiRNA-mediated Lurap1 knockdown in A549 and H1299 cell lines showed the opposite results.5.Lurap1 inhibited EMT of NSCLC cells.We found that the expression of E-cad and ZO1 were up-regulated,and the expression of N-cad was down regulated when Lurap1 was overexpressed in A549 cell lines.SiRNA-mediated Lurap1 knockdown in A549 cell lines showed the opposite results.6.Lurap1 can interact with Dvl1,2 and 3 in NSCLC cells.We found that the overexpression of lurap1 could bind to DVL1,2 and 3,respectively when Lurap1 was overexpressed in A549 and H1299 cell lines.Conclusion : 1.The expression of Lurap1 is low in NSCLC cytoplasm,which is related to TNM stage and lymph node metastasis in NSCLC patients.2.Overexpression of Lurap1 can inhibit the proliferation and invasion of NSCLC cells.3.Lurap1 inhibits the proliferation and invasion of NSCLC cells by inhibiting Wnt pathway and activating Hippo pathway.4.In NSCLC,lurap1 can interact with DVL1,2 and 3.