The Promotion Of Lung Adenocarcinoma Metastasis By L1CAM And The Underlying Regulatory Mechanism

Objective:To clarify the effect of L1CAM(L1 cell adhesion molecule)on the proliferation,migration and invasion of lung adenocarcinoma cells,and to reveal the molecular mechanism regulating L1 CAM function.Method:1.Clinical relevance of L1 CAM and lung adenocarcinoma and its biological function in tumors Collect 88 cancerous and adjacent tissue samples from matched lung adenocarcinoma patients,and use immunohistochemical techniques to detect the expression level of L1 CAM,and analyze the clinical correlation between the expression level and the prognosis of the patients.2.The effect of LICAM on the proliferation,invasion and migration of lung adenocarcinoma cells(1)L1CAM was overexpressed or silenced in lung adenocarcinoma cell lines A549,NCI-H1299,and NCI-H1770,and L1 CAM was used to verify the proliferation,invasion,and migration ability of lung adenocarcinoma cells through Transwell,cell proliferation,and Wound healing.(2)The mouse endotrachial orthotopic tumor model of sh L1 CAM was constructed by the transposition system.The mice were sacrificed 6 weeks after injection,and lung tissue specimens were taken to count the number of tumors formed.(3)L1CAM was overexpressed or silenced in H460 SM cells,and an orthotopic tumor model in nude was established by in situ inoculation via bronchial lungs.Nude were sacrificed after reaching the expected endpoint,and tumor size and metastasis were compared between the experimental group and the control group.3.Identify the pathways that up-regulate L1 CAM expression in lung adenocarcinoma and their effects on biological functions(1)Detect the expression of L1 CAM after silencing PAK4 and Slug by si RNA interference technology and Western blot in H1770 cells;(2)The protein expression levels of PAK4 and Slug were verified by Western blot in the previously collected surgical samples of lung cancer patients;(3)Explore mi RNAs that inhibit PAK4 through bioinformatics analysis;(4)Transfection of mi RNA or its inhibitors by RNAi technology in SPC-A-1,SPC-A-1sci,using q PCR,Western blot experiments to detect PAK4,Slug,L1 CAM expression and EMT-related proteins(E-cadherin and Vimentin)Expression level.(5)Transwell and Wound healing assay were used to verify the ability of SPC-A-1and SPC-A-1sci cells to invade and migrate after transfection.(6)In nude,the tail vein was injected with SPC-A-1sci or SPC-A-1 cells after transfection,and the number of lung metastases in mice was measured 7 weeks later to further verify that mi RNAs in lung adenocarcinoma through the PAK4/Slug/L1 CAM pathway biology functions.Results:1.In the collected specimens of lung adenocarcinoma,the expression level of L1 CAM in cancer tissues was higher than that in the adjacent tissues.The survival analysis showed that the L1 CAM high expression group had a worse prognosis than the low expression group(P <0.05).2.After overexpression of L1 CAM in A549,NCI-H1299,and NCI-H1770 cell lines,the lung adenocarcinoma cell invasion and migration ability was significantly enhanced compared with the control group;silence L1 CAM,the above three groups of lung adenocarcinoma cell invasion and migration ability were significant Reduced,and can inhibit the occurrence of primary lung cancer and the number of metastatic tumors in mouse tumor models.However,the results of cell proliferation experiments showed that there was no significant difference in cell proliferation ability after knockdown or overexpressing L1 CAM compared with the control group.3.In tumor tissue samples,PAK4,p-Slug and L1 CAM protein levels were higher in patients with tumor metastasis than in patients without metastasis.Bioinformatics analysis showed that mi R-193a-3p binds to 3'-UTR of Slug's activator PAK4.It was confirmed by q PCR and Western blot experiments that mi RNAs inhibited PAK4 m RNA,the expression levels of p-Slug and L1 CAM decreased;while anti-193a-3p transfected SPC-A-1 upregulated the expression of p-Slug and L1 CAM.4.In SPC-A-1 cells transfected with anti-193a-3p,the expression level of E-cadherin protein in SPC-A-1 cells was lower than that in the control group,and the expression level of vimentin in interstitial cell marker was increased,Suggesting that anti-193a-3p promotes the EMT process of the transfected cells;while the SPC-A-1sci cells transfected with mi R-193a-3p analogs have increased E-cadherin protein expression levels and decreased Vimentin protein expression compared with the control group Prompted that the EMT process was suppressed.5.In the Wound healing assay,the migration ability of SPC-A-1sci cells was significantly reduced;the migration ability of SPC-A-1 cells was significantly increased compared with the control group.6.In nude,the transvenous SPC-A-1 cell line was injected into the tail vein,and the number of metastatic tumor nodules was significantly less in the lung tissue.While the lung tissue of the nude in the anti-193a-3p group was compared with other control groups,has more metastatic nodules(P <0.01).Conclusion:1.The high expression of L1 CAM is closely related to the survival rate of lung adenocarcinoma patients;2.The expression level of L1 CAM is positively correlated with the ability of lung adenocarcinoma to migrate and invasion,but has no significant effect on the tumor proliferation ability;3.PAK4 regulates the PAK4 / Slug / L1 CAM pathway by phosphorylating Slug and promotes L1 CAM expression;mi R-193a-3p regulates the PAK4 / Slug / L1CAM pathway by binding to PAK4 m RNA,thereby inhibiting the EMT and migration /invasion of lung adenocarcinoma ability.