Reversal Effect And Mechanism Of β-Elemene On The Promotion Of Migration And Invasion By ALX1 Via EMT Induction In Lung Cancer Cells

Part I Expression of ALX1 in lung cancerObjective: To study the expression and significance of ALX1 in lung cancer, and to investigate the relationship between ALX1 expression and lung cancer malignancy and prognosis.Methods: Real time-PCR and immunohistochemistry were conducted to study the expression levels of ALX1 in 47 lung cancer samples. Kaplan-Meier survival analysis was used to investigate the relavance of ALX1 expression and lung cancer prognosis in clinic.Results: 1. m RNA levels of ALX1 were higher in tumors than that in adjacent tissue. 2. In tumors, ALX1 m RNA expressed higher in metastatic lung cancer than that in non-metastatic lung cancer. 3. Immunohistochemistry assays showed that a high level of ALX1 expression was significantly more common in metastatic lung cancer than that in non-metastatic lung cancer. 4. Kaplan-Meier survival analysis showed significant survival extension of ALX1-low expression lung cancer compared with ALX1-high expression lung cancer.Conclusion: Patients with higher ALX1 expression levels had poorer disease free survival(DFS) than those with lower ALX1 expression levels in lung cancer. ALX1 significantly correlated with prognosis of lung cancers.Part II Construction, identification and application of lung cancer cells transfected with ALX1 or si RNA ALX1Objective: To construct lung cancer cells with ALX1 overexpression or silence, and to reveal the role and molecular mechanisms of ALX1 in migration and invasion of lung cancer cells.Methods: Human gene ALX1 was cloned into p Babe-puro vector and sh RNA(1, 2 and 3) of ALX1 was cloned into p Super-puro vector. The levels of ALX1 expression were determined by Western blot to validate the models. MTT assays were conducted to investigate lung cancer cell proliferation. Western blot and immunofluorescence were conducted to investigate EMT induced by ALX1 in lung cancer cells.Results: 1. ALX1 expression levels in 8 lung cancer lines were different. H1975 cells with low level and H460 cells with high level were selected for the transfected study. 2. After transfected, protein expression levels of ALX1-p Babe-H1975 were higher than that in corresponding control cells; and ALX1-si RNA-H460 showed a low level of protein expression. 3. Overexpression of ALX1 significantly promoted H1975 cell proliferation while silencing ALX1 inhibited the proliferative rate of H460 cell. 4. Under microscope, H1975 cell overexpressing ALX1 showed mesenchymal morphology compared with control cell while H460 cell silencing ALX1 showed epithelial morphology compared with control cell. 5. Downregulation of epithelial cell marker(E-cadherin and alpha-catenin) and upregulation of mesenchymal cell markers(vimentin and N-cadherin) were observed by Western blot in ALX1 overexpressing cell while the opposite expressions of EMT markers were found in ALX1 silencing cell. The same results were found through immunofluorescence analysis.Conclusion: ALX1-p Babe-H1975 and ALX1-si RNA-H460 cell models were constructed successfully and applied in the study of role of ALX1 in the EMT induction in lung cancer cells.Part III Mechanism of ALX1 on the EMT induction in lung cancer cellsObjective: To investigate the role and mechanism of ALX1 on the EMT induction in lung cancer cells.Methods: Transwell and matrigel assays, Real time-PCR experiments, Western blot and luciferase assay were conducted using ALX1 overexpression and knockdown cell models.Results: 1. Overexpression of ALX1 in H1975 cell significantly promoted more cells migrated or invaded through the membrane to the bottom of the aperture while silencing ALX1 in H460 restrained these progresses. 2. The RNA level and the protein level of Snail increased by the overexpression of ALX1 in H1975 cell, while both the RNA level and protein level of Snail decreased by exogenous sh RNA of ALX1 in H460 cell. Expression levels of ZEB1, Slug and Twist were not changed by overexpression or knockdown of ALX1. 3. ALX1 significantly increased the intensity of luciferase in H1975 cell while the decreases of luciferase intensity were examined in ALX1 silencing cells. 4. Knockdown of Snail in H1957-p Babe-ALX1 cell restored the ALX1-induced downregulation of E-cadherin and alpha-catenin and upregulation of Vimentin and N-cadherin. Knockdown of Snail restored the increasing numbers of cells migrating through the membrane to the bottom of the aperture in transwell assay as well as in matrigel assay.Conclusion: ALX1 induced EMT in lung cancer cells via upregulation of Snail, promoting migration and invasion. These findings may serve as a framework for future investigations designed to more comprehensive determination of ALX1 as a potential therapeutic target.Part IV Reversal effect and mechanism of β-elemene on EMT induced by ALX1 in lung cancer cellsObjective: To investigate the effect and mechanism of β-elemene on ALX1-induced EMT in lung cancer cells.Methods: MTT assays, Transwell and matrigel assays, immunofluorescence and Western blot were conducted using ALX1 overexpression cell models.Results: 1. β-elemene treatment inhibited proliferation of ALX1-p Babe-H1975 cells and changed the cell morphology. 2. β-elemene treatment significantly attenuated migration and invasion of ALX1-p Babe-H1975 cells through the membrane to the bottom of the aperture. 3. Upregulation of epithelial cell marker(E-cadherin and alpha-catenin) and downregulation of mesenchymal cell markers(vimentin and N-cadherin) were observed incubation of ALX1 overexpressing cell with β-elemene. Immunofluorescence analysis increased intensity of E-cadherin and decreased intensity of N-cadherin. 4. The protein level of Snail was decreased by β-elemene.Conclusion: β-elemene inhibited the proliferation, migration and invasion of ALX1 overexpressing cell via Snail inhibition. β-elemene can be used as a potential drug treatment of lung cancer with overexpression of ALX1.