The Expression Of Lyn In Lung Adenocarcinoma And The Effects Of Overexpression Of Lyn On The Biological Behavior Of A549 Cells

Background:The morbidity and mortality of the Lung cancer is located in the first place in China,which is called "the king of cancer",its incidence is still increasing and showing a trend of younger.With the continuous development of China’s industrial technology,environmental pollution is becoming increasingly serious,now lung adenocarcinoma has replaced the lung squamous carcinoma as the most common histological type of lung cancer.Lung adenocarcinoma was always found in the late stage,and the treatment of radiotherapy and chemotherapy in lung cancer is attended with poor results,which result in the prognosis of lung adenocarcinoma is not ideal.So exploring with lung adenocarcinoma occurrence and development associated genes has important significance.Research has shown the high level of Lyn in leukemia,lymphoma,prostate cancer,colon cancer and breast cancer,which was one major member of the Src family of intracellular menbrane-associated kinases(SFK).Lyn also regulates activation of epidermal growth factor receptors in lung adenicarcinoma cells.However,there are no more reports about the expression of Lyn in lung adenocarcinoma tissues,the relationship between the clinicopathological features and the expression of Lyn in lung adenocarcinoma still need further study.Objective:To detect the expression of Lyn in human lung adenocarcinoma and its adjacent noncancerous tissues,Analysis the relationship between the abnormal expression of Lyn and clinical pathological features of lung adenocarcinoma,and then evaluate the clinical significance of abnormal expression of Lyn in lung adenocarcinoma tissues.human lung adenocarcinoma cells A549 were transfected with virus which carrying Lyn,to investigate the effect of high expression of Lyn on the proliferation,migration and invasion of the Lung adenocarcinoma cells A549,and further study the relationship between the abnormal expression of Lyn and biological behavior of lung adenocarcinoma cells.this experiment is divided into the following 2 parts:Part Ⅰ The Expression and Significance of Lyn in lung adenocarcinomaMethods:1)Approved by the ethics committee of Affiliated Hospital of Luzhou Medical College,38 cases of lung adenocarcinoma which have complete clinical data were collected in cardiothoracic surgery of Affiliated Hospital of Luzhou Medical College from March 2015 to September 2015.38 cases of lung adjacent tissues were collected at the same time.A part of the tissues are to be used for making frozen sections,and the frozen sections were stored in the-70℃ fridge,another part of the tissues are also stored in the-70℃ fridge for extracting total RNA and total protein.2)Extracting the total RNA and total protein of lung adenocarcinoma and adjacent tissues,RT-PCR was used to detect the expression of Lyn m RNA in lung adenocarcinoma and adjacent tissues,Western blot and immunofluorescence was used to detect the expression of Lyn protein in lung adenocarcinoma and adjacent tissues.Analysis the relationship between the abnormal expression of Lyn and clinical pathological features of lung adenocarcinoma,and then evaluate the clinical significance of abnormal expression of Lyn in lung adenocarcinoma tissues.Results:1)The results of RT-PCR showed that compared with the adjacent tissues,the expression of Lyn m RNA in lung adenocarcinoma tissues was higher,the difference was statistically significant(P=0.005,P < 0.01).2)Immunofluorescence and Western blot indicated that the expression of Lyn protein in lung adenocarcinoma tissue are higher than adjacent tissue,the difference was statistically significant(P < 0.05).3)Through the analysis and comparison of clinical pathological data showed that the expression level of Lyn was not related with gender,age,smoking history,but it was related with clinical stage and lymph node metastasis(P<0.05).Conclusions:Lyn was highly expressed in lung adenocarcinoma.The high expression of Lyn was not related with gender,age and smoking history,but it was related with clinical stage and lymph node metastasis,suggesting that Lyn is involved in the invasion and metastasis of lung adenocarcinoma,and Lyn may be participated in the occurrence and development of lung adenocarcinoma.Part Ⅱ Effects of overexpression of Lyn on the biological behavior of A549 cellsMethods:1)Human lung adenocarcinoma cells A549 were transfected with virus which carrying Lyn,non-Lyn virus transfect lung adenocarcinoma A549 cells as control group,using the confocal microscope to observe the transfection efficiency,and to obtain A549 which stably overexpress Lyn by puromycin screening.2)RT-PCR was used to detect the expression of Lyn m RNA in A549,A549/GFP,A549/Lyn cells,immunofluorescence was used to detect the expression of Lyn protein in each group cells,to verify the transfection efficiency further.3)Using MTT method to detect the living cells of A549,A549/GFP and A549/Lyn cells at different time points,to evaluate the proliferation ability of each group cells.4)Vitro invasion assay(Transwell)was used to study the invasiveness of A549,A549/GFP and A549/Lyn cells in vitro.Results:1)Green fluorescence was observed in transfected cells by confocal microscopy,and the number of living cells with Spontaneous green fluorescence is more than 90%,indicating that the virus was successfully transfected.2)RT-PCR and immunofluorescence results showed that Compared with A549 and A549/GFP cells,the expression of Lyn m RNA and Lyn protein in A549/Lyn cells were increased,differences were statistically significant(P < 0.05).3)The results of MTT assay showed that compared with A549 and A549/GFP cells,the proliferation of A549/Lyn cells were significantly increased,the difference was statistically significant(P<0.05).4)Transwell assays results showed that the invasive ability of A549/Lyn cells are higher than A549 and A549/GFP cells,the difference was statistically significant(P < 0.05).Conclusions:1)The virus which carrying Lyn transfect human lung adenocarcinoma cell line A549 could successfully obtained A549 cells that overexpress Lyn.2)Lyn could significantly enhance the proliferation and invasion of Human lung adenocarcinoma cell A549.Lyn is expected to become the new therapeutic targets of lung adenocarcinoma patients.