Functional MUC4Suppress Epithelial-mesenchymal Transition In Lung Adenocarcinoma Metastasis

ObjectiveLung cancer is a major cause of cancer death, and lung adenocacinoma is the most common type of lung cancer. MUC4mucin, which is a high-molecular-weight membrane-bound trans-membrane glycoprotein, is associated with risks of several kinds of cancer, and overexpression of MUC4is frequently detected in invasiveness and metastatic cancaer. Epithelial-mesenchymal transition, is a highly conserved cellular process in which cells undergo a developmental switch from polarized generally immotile epithelial cells to motile mensenchymal-appearing cells, it exists in animals multiple physiological and pathological process, and involves complex signal path adjustment process, performs a variety of physiological functions.This process was initially described during early embryonic development and has been implicated in relating to fibrosis, and lately,to the invasion and metastasis of malignant tumor. Our research was to study the functional role of MUC4associated with invasion and metastasis, meanwhile, we further investigated its regulatory mechanisms epithelial-mesenchymal transition in lung adenocacinoma, providing new targets for diagnosis and treatment of lung adenocarcinoma.MethodsWe routinely cultured lung adenocarcinoma cell lines, Western blotting was used to detect the expression of mucin MUC4in lung adenocarcinoma cell lines. We constructed MUC4-siRNA expression vector to transfect lung adenocarcinoma cell lines by Iiposomal2000, divided into experimental group (transfected with vectors MUC4-siRNA) and control group (empty vector MUC4-siRNA),Western blotting was used to detect the expression of MUC4, E-cadherin and Vimentin; Inverted microscope was used to observe the changes of cell morphology; In vitro migration assay, we investigated the relationship between MUC4and the level of cell migration; Immunofluorescence microscopy assay was used to observe the impact of MUC4on β-catenin, meanwhile, quantitative Western Blotting assay was used to observe the expression of β-catenin in the cell membrane, cytoplasm and nucleus. Animal experiments was divided two groups:MUC4-knockdown-LTEP planting rat (129/sv) and MUC4-control-LTEP planting rat (129/sv). Immunohistochemistry and Western blotting assay was used to observe the expression of MUC4, E-cadherin and Vimentin in the lung tissue of two groups mice. We respectively screened15cases of patients with stage I and III from Tianjin Cancer Hospital through PET-CT, meanwhile, collected lung tissue specimens, the pathologic result confirmed that specimens from patients with stage I and III was respectively10cases of lung adenocarcinoma specimens. Western Blotting assay was used to detect the expression of mucin MUC4in20cases of lung cancer tissue specimens. We statistically analyzed the expression of mucin MUC4in20cases of lung cancer tissue specimens, investigated the relationship between the level of mucin MUC4and lymph node metastasis in lung adenocacinoma patients.Resultsl.In vitro cell assay, mucin MUC4expressed highly in the lung adenocarcinoma cell line LTEP and SPCA, however, the expression of mucin MUC4was low in the lung adenocarcinoma cell line GLC-82and A549. We transfected LTEP cell with MUC4-siRNA transfection vectors by liposomal2000, LTEP cells undergone epithelial-mesenchymal transition, compared with the control group, the expression of mucin MUC4and epithelial marker E-cadherin was reduced, however, expression of mesenchymal marker Vimentin was increased, LTEP cells showed a distinct phenotypic transition from cobblestone-like cells(transfected with control-MUC4) to spindle-like cells (transfected with MUC4-siRNA), the cells also showed disordered cell polarity and cellular contacts.2.In vitro migration assay, compared with the control group, the number of migratory cells showed a significant (P<0.01) increase in LTEP cells (transfected with MUC4-siRNA).3.1mmunofluorescence microscopy assay and quantitative Western Blotting assay: compared with the control group, in LTEP cells (transfected with MUC4-siRNA),β-catenin showed the accumulation of cytoplasm and transferred to nuclear, however, β-catenin binding to the cell membrane was reduced.4.Orthotopic animal model:compared with MUC4-control-LTEP planting rat (129/sv), in MUC4-knockdown-LTEP planting rat (129/sv) tumor lung tissue, the expression of mucin MUC4and epithelial marker E-cadherin was significantly reduced, however, expression of mesenchymal marker Vimentin was increased.5.Quantitative Western Blotting assay in human lung adenocarcinoma specimens: we found that MUC4low expression existed in6of10specimens without lymph node metastasis, however, in ten specimens with lymph node metastasis, all showed MUC4low expression. We analyzed the clinico-pathological data (n=20) of MUC4expression and lymph node metastasis statistically. The data showed that MUC4low expression was relate to lymph node metastasis (p<0.01).Conclusions1.Low expression of mucin MUC4induced LTEP cells to epithelial-mesenchymal transition.2.With the Low expression of mucin MUC4, the number of migratory cells showed a significant (P<0.01) increase in LTEP cells (transfected with MUC4-siRNA). These date indicated that mucin MUC4was associated with invasion and migration in lung adenocarcinoma cell.3.With the Low expression of mucin MUC4, in LTEP cells (transfected with MUC4-siRNA), β-catenin showed the accumulation of cytoplasm and transferred to nuclear, however,β-catenin binding to the cell membrane was reduced, we found that MUC4modulated β-catenin nuclear translocation to repress epithelial-mesenchymal transition in lung adenocarcinoma cell lines.4.Through orthotopic animal model, we found the expression of mucin MUC4and epithelial marker E-cadherin was significantly reduced, however, expression of mesenchymal marker Vimentin was increased in MUC4-knockdown-LTEP planting rat (129/sv) tumor lung tissue. This showed mucin MUC4expression was related with epithelial-mesenchymal transition in vivo assay.5.MUC4low expression existed in6of10specimens (TNM staging I)without lymph node metastasis, however, in ten specimens(TNM staging Ⅲ) with lymph node metastasis, all showed MUC4low expression. Through statistical analysis showed that MUC4low expression was relate to lymph node metastasis (p<0.01), these results obviously illustrated that MUC4expression had a close relationship with lung adenocacinoma invasion and metastasis. Therefore, the assessment of MUC4may function as a prognostic biomarker and could be a potential therapeutic target for lung adenocarcinoma metastasis.