| BackgroundLung cancer is one of the most common malignant tumors in the world. The morbidity and mortality of lung cancer is increasing every year,and it is the leading cause of cancer-related death in many countries. In China, the incidence of lung cancer and the related mortality has significantly increased in recent years.Approximately 80–85% of lung cancer are non-small cell lung cancer,which mainly includes squamous cell carcinoma,adenocarcinoma,large cell carcinoma and so on.Surgical treatment is still the first choice for early stage non-small cell lung cancer,which conbined with postoperative chemotherapy and biological treatment, has significantly improved five year survival rate. However,about 50% of lung cancer patients are diagnosed as advanced stage and lose the opportunity for surgery. For these cases,chemotherapy and radiation therapy are the primary treatment. However,effectiveness of these therapeutic methods,which present severe side effects,has nearly reached a plateau. Therefore,molecular targeted therapy,which offers a great deal of specificity and promise minimal side effects, now has become the most important treatment for lung cancer.In recent years,more and more molecular targets are discovered for targeted therapy in malignant tumor,such as epidermal growth factor receptor(EGFR),vascular endothelial growth factor receptor(VEGFR),Her2,ROS1 and so on.Many targeted drugs are emerging,part of which has been approved for clinical application,espically for those advanecd stage NSCLC. Targeted therapy has achieved considerable efficacy in clinic,prolonged the five year survival rate of some advanced cases,and led to a new era of individualized treatment in lung cancer. Therefore,further exploration of new molrcular targets plays very important role for lung cancer treatment.Epithelial-mesenchymal transition(EMT) refers to epithelial cells lose the epithelial characteristics,obtain the mesenchymal cell characteristics,and achieve the ability of degradating extracellar matrix,migration and invasion.Many studies have demonstrated that EMT is a key process related to aggressive epithelial-derived cancer progression,and it is the first step for migration and invasion in maglinant tumors.Nowadays, EMT has become an important target for the research of anti-tumor drugs. Transforming growth factor(TGF-β)signal pathway is considered to be the most ralated signal pathway for EMT.Nodal protain,which belongs to this signal superfamily,is essential for supporting an undifferentiated state in embryonic stem(ES)cells and for endodermal and mesodermal differentiation,including EMT.Recently, Nodal expression has been detected in a number of malignant tumors,including melanoma, breast,pancreas and prostate cancer. Further researches have discovered that Nodal participates in malignant tumor proliferation, invasion and metastasis, and affect their prognosis.Above all, Nodal is likely to be a new molecular target for targeted therapy in malignant tumors.At present,the research of Nodal in lung cancer is rare.To discover whether Nodal expresses in lung cancer,we started our study from the following aspects:(1)NSCLC specimens were collected,immunohistochemistry was used to detect Nodal expression,and the correlation between Nodal expression and clinical related factors was analyzed;(2)lung cancer cell line A549 were cultured,immunocytochemistry and Western blot were used to detect Nodal expression in A549;(3)In the end,we studied the related mechanism of Nodal induced metastasis in Non-small cell lung caner by vitro cell experiments.Part Ⅰ Nodal expression in non-small cell lung cancer and clinical significanceObjectiveTo detect Nodal expression in human non-small cell lung cancer and analyze its clinial significance.Methods1. Nodal protain was detected in 24 cases of squamous cell carcinoma and 70 cases of adenocarcinoma by immunohistochemical staining. Twenty cases of nornal lung tissue was used for control group.2. Analyze the correlation of Nodal expression between clinical factors such as age,sex,smoking history,histological type and differentiation,clinical stage,lymph node metastasis and oligometastasis.ResultsImmunohistochemical staining showed Nodal positive expression rates of squamous cell lung cancer and lung adenocarcinoma were 33.3%(8/24) and 84.3%(59/70)respectively. Nodal expression was significantly higher than control group whose positive expression rates was 10.0%(p<0.05), but there was no statistical difference between squamous cell lung cancer and normal lung tissues(p>0.05).Nodal positive expression rate in high differentiation lung adenocarcinoma(76.7%)was lower than low differentiation(96.3%)(p<0.05). Nodal positive expression rates in clinical stage III-IV(94.7%) was higher than I-II(71.9%)(p<0.05). For patients with lymph node metastasis,Nodal positive expression rate was 92.8%,which was higher than without lymph node metastasis(71.4%)(p<0.05). For patients with and without oligometastasis,Nodal positive expression rates were 96.2% and 77.3% respectively,and there was statistical difference between them(p<0.05).There were 39 male and 31 female cases in lung adenocarcinoma,Nodal positive expression rates were 89.8% and 77.4% respectively,and no significant difference was found(p>0.05). Nodal expression was found no statistical difference between patients who were less than 50 years(85.7%)and over 50 years( 83.7%)(p>0.05).Nodal positive expression rates rates in patients with and without smoking history were 76% and 88.9% respectively,which shows no statistical difference(p>0.05).ConclusionNodal positive expression rate in lung adenocarcinoma was significantly higher than normal lung tissue,but there is no significant difference between squamous cell carcinoma and normal lung tissue. In lung adenocarcinoma,Nodal expression has obvious relationship with differentiation degree,node metastasis,distant metastasis and clinical stage. Nodal positive expression rate was detected higher in those patients with lower differentiation degree,advanced clinical stage,lymph node metastasis and distant metastasis,but there is no relationship with age,sex or smoking history.Part Ⅱ Nodal expression in lung adenocarcinoma cell line A549ObjectiveTo culture lung adenocarcinoma cell line A549, and detect Nodal protein expression.Methods1. To recovery,culture and subculture lung adenocarcinoma cell line A549.2. Nodal protein was detected by immunocytochemical staining and Western blot in subcultured A549,normal alveolar epithelial cell was used for control group.ResultsThe A549 cells were large and polygonal,displaying the typical cobblestone appearance. Brown medium positive staining granules(++)in cytoplasm of A549 were detected by immunocytochemical staining,but negative staining was found in normal alveolar epithelial cell. Western blot further dectected the expression of Nodal protein was significantly higher than control group.ConclusionNodal expression was positive in lung adenocarcinoma cell line A549, but negative in normal alveolar epithelial cell.Part Ⅲ The mechanism in Nodal affect on non-small cell lung cancer metastasisObjectiveTo explore the mechanism in Nodal affect on lung adenocarcinoma invasion and metastasis.Methods1. Recombinant mouse Nodal protein(r Nodal) was used to intervene subcultured A549 cells.To observe the r Nodal induced morphological changes with microscope.2. To collect the r Nodal intervened A549 cells which were cultured to 12 h,24h and48 h,extract total protein in each period. The EMT markers of E-cadherin and Vimentin protein expression of different time periods were detected by Western blot.3. Cell scratch test and Transwell migration test experiments were used to observe r Nodal effect on the migration and invasion of A549 cell line.4. To collect the r Nodal intervened A549 cells which were cultured to 15,30 and60 min,extract total protein in each period.The Smad2 phosphorylation levels at different time periods were also detected by Western blot.ResultsThe normal A549 cells were large and polygonal,displaying the typical cobblestone appearance,when treated with r Nodal,the cells were gradually became irregular and spindle shaped at 24 h,and most obvious at 48 h,which had time dependence. E-cadherin protein expression decreased with the r Nodal intervened time,but Vimentin protein expression was on the contrary,which showed an upward tendency.The migration cells were observed in the scratch area after 24 hours of scratch test.The number of migration cells was the largest in the r Nodal intervened group at 48 h,but the least in the r Nodal inhibitor SB group.The number of migration cells in r Nodal intervened group was significantly larger than control group.In Transwell invasion assay,the number of invasive tumor cells passed through the Matrigel membrane in r Nodal intervened group was significantly larger than control group and r Nodal inhibitor SB group(p<0.05).ConclusionNodal can induce epithelial-mesenchymal transition(EMT) in A549 cell line,which included the morphological changes(time-dependent), the EMT markers of E-cadherin and Vimentin protein changed.Nodal can also promote the migration and invasion ability of A549 cells. |
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