Mechanism Study Of CSMD3 In The Occurrence And Development Of Non-small Cell Lung Cancer

Background: Lung cancer is a malignant tumor that seriously threatens human life and health.It is one of the leading causes of cancer-related death,causing 1.6 million deaths every year,among which non-small cell lung cancer(NSCLC)accounts for 80% of the total number of lung cancer,including lung adenocarcinoma and squamous cell carcinoma.Non-small cell lung cancer(NSCLC)is the largest cancer in men and the second largest cancer in women among cancer patients.So far,especially in the past 20 years,the application of various targeted therapies and immunotherapy has been extending the survival period of lung cancer patients.CSMD3(CUB and Sushi multiple domains 3)is a newly discovered tumor suppressor gene in the 21 st century,located on human genome chromosome 8.It has been confirmed that CSMD3 is expressed in nervous system,lung,liver,spleen and other organs.At the same time,more and more studies have found that abnormal expression of CSMD3 is closely related to the occurrence and development of various diseases,especially cancer.For example,gene deletion of CSMD3 in head and neck squamous cell carcinoma(HNSCC),breast cancer and lung cancer is a common phenomenon,and there is a significant correlation between CSMD3 and the development and prognosis of HNSCC.Low expression of CSMD3 protein is closely related to tumor differentiation,lymph node metastasis and tumor size.The results of this study coincide with the findings of this study in 20 pairs of lung adenocarcinoma samples.Past for CSMD3 research in lung cancer more than several in the correlation research level,for CSMD3 is how to affect the occurrence and development of lung cancer is still unknown,this study from CSMD3 as the breakthrough point,to CSMD3 expression in lung cancer tissue and normal tissue expression differences,TCGA database CSMD3 and survival of patients with lung cancer,etc correlation research and further confirmed that the next for CSMD3 through what way affects the occurrence and development of lung adenocarcinoma,this study will from the organization's level and cell level in-depth and detailed discussion and research,It is believed that this study will provide basic data and theoretical support for the early diagnosis and prognosis of lung adenocarcinoma and the development of new targeted therapies.Part ?: Mechanism study of CSMD3 the occurrence and developmentlung adenocarcinomaObjective: To study the expression changes of CSMD3 in lung adenocarcinoma tissue samples and A549 cell lines,and to understand the effects of CSMD3 on the capacity and function of lung adenocarcinoma cell lines A549,as well as the effects on the downstream gene expression levels when the expression level of CSMD3 is down-regulated.Methods: In this study,real-time PCR and Western Blot were first used to study the expression level of CSMD3 in the cancer-adjacent tissue samples collected from 20 patients with lung adenocarcinoma,respectively at the transcription level and translation level.The correlation between CSMD3 expression and survival of patients with lung adenocarcinoma was studied using TCGA database.Next,this study constructed the knockdown viral vector of CSMD3 and transfected the A549 cell line.The migration ability of the A549 cell line was studied by using the cell scratch test to explore the effect of CSMD3 on the migration ability of the A549 cell line.The activity of A549 cell line knocked down by CSMD3 was studied by using ccck-8 technology to explore the effect of CSMD3 on the proliferation capacity of A549 cell line.Subsequently,this study also used the transwell chamber to study the invasiveness of A549 cell lines under the condition of CSMD3.Finally,in this part,human gene expression profile chip was used to study genes with differential expression under CSMD3 knockdown conditions,and the differentially expressed genes were verified by real-time PCR.Results: 1.In the detection of 20 lung adenocarcinoma cancer-paracancerous tissue samples,it was found that the expression of CSMD3 gene in the cancer tissues was lower than that in the paracancerous tissues regardless of the transcription level or the protein level.2.Analysis of the data of lung adenocarcinoma in the TCGA database found that the expression of CSMD3 had no significant correlation with the survival of patients with lung adenocarcinoma.3.The proliferation,migration and invasion ability of the A549 lung adenocarcinoma cell lines of CSMD3 were significantly improved.4.In the detection of human expression profile chip,a total of 887 gene expression changes were found in A549 lung adenocarcinoma cell lines with artificial knockdown of CSMD3 compared with the control group,including 653 up-regulated genes.According to the results of cluster analysis,the functions of differentially expressed genes mainly include cancer,cell movement,injury and disorder,cell death and survival,and cell growth and proliferation.Conclusions: Although there is no significant correlation between CSMD3 and survival of patients with lung adenocarcinoma,data in this study showed that the expression level of CSMD3 in cancer-adjacent tissues was significantly different,and was closely related to the proliferation,migration and invasion capacity of A549 cell lines,suggesting that CSMD3 may indirectly be involved in the occurrence and development of lung adenocarcinoma.At the same time,this study found that the artificial knockdown of CSMD3 resulted in the increased expression of two cancer-related genes,TCTP and PLOD2.In the following part,we will conduct in-depth study on how CSMD3 affects the occurrence and development of lung adenocarcinoma through the above two genes.Part ?: The effect TCTP affects the function of epithelial interstitial transformation of lung adenocarcinoma A549 cell lines through CSMD3Objective: In the last part of the study,we found that the downregulation of CSMD3 can increase the proliferation and invasion of A549 cell,and CSMD3 can lead to significantly up-regulated TCTP gene expression.To further investigate how CSMD3 affects the migration and metastasis of A549 cell lines through TCTP.To explore the effect of TCTP on A549 cells when expression level is up-regulated or down-regulated.Methods: First,tissue samples of 20 patients with lung adenocarcinoma collected in this study were used to study the correlation between TCTP gene expression in cancer-adjacent tissues,tumor stage and tumor size,and TCGA database was used to explore the correlation between TCTP expression level and patient survival.Next,this study constructed TCTP gene knockdown and overexpression lentiviral vector and transfected A549 cell lines.After verifying TCTP knockdown and overexpression efficiency,the migration and metastasis capacity of TCTP in the case of knockdown and overexpression were studied using the cell scratch experiment and cell invasion experiment respectively.At the same time,Western Blot and immunofluorescence staining were used in this study to study the expression of the marker protein of epithelial interstitial transformation in the A549 cell lines of lung adenocarcinoma A549 cells: ?-SMA,ZEB1,e-cadherin and P53,respectively,in the case of TCTP knockdown and overexpression.In addition,we also used real-time PCR to study the expression of mir-200 family in the TCTP knockdown(overexpression)A549 cell line,in order to explore how changes in TCTP expression affect the epithelial interstitial transformation of lung adenocarcinoma A549 cell line.Results: 1.In the detection of 20 lung adenocarcinoma cancer-adjacent tissue samples,it was found that the expression of TCTP gene in the cancer tissue was higher than that in the adjacent tissue regardless of the transcription level or the protein level.2.Analysis of the data of lung adenocarcinoma in the TCGA database found that the expression level of TCTP was significantly correlated with the survival period of lung adenocarcinoma patients(P<0.01),where the median survival period of the low expression group was 78.5 months,and the median survival period of the high expression group was only 61.2 months,that is,the survival period of patients with high expression of TCTP was short.3.In A549 lung adenocarcinoma cells with TCTP knockdown,TCTP knockdown led to a significant reduction in migration efficiency compared with the control group in the scratch test(P<0.05).Meanwhile,compared with the control group,TCTP knockdown can significantly reduce the number of cells passing through the transwell chamber(P<0.05).4.Compared with the control group,the expression level of the marker protein e-cadherin of the A549 cell line of TCTP was increased,while the ?-SMAexpressionwas significantly decreased.In addition,the expression level of the tumor suppressor gene P53 was significantly increased,suggesting that TCTP knockout could inhibit the transformation process of the A549 cell line of the epithelium.5.Overexpression of TCTP in A549 cell lines resulted in a significant increase in migration efficiency in scratch test(P<0.05).Meanwhile,compared with the control group,overexpression of TCTP significantly increased the number of cells passing through transwell cells(P<0.05).6.In the A549 cell lines overexpressing TCTP,the expression level of e-cadherin,the marker protein of the epithelium,was lower than control,while the expression level of the marker protein ?-SMA and ZEB1 was significantly increased.In addition,the expression level of the tumor suppressor gene P53 was significantly reducd,suggesting that the overexpression of TCTP can promote the transformation process of the A549 cell lines.7.The expression level of mir-200 s family was down-regulated in the TCTP overexpressed A549 cell line,while the expression level was up-regulated in the TCTP knockdown A549 cell line.Conclusions: In the TCTP gene expression of A549 cell line,the migration and invasion ability had occurred significantly increased,moreover epithelial marker protein E-cadherin expression quantity,and interstitial cell marker protein a-SMA and ZEB1 expression quantity to increase,at the same time,the expression quantity of mi R-200-s family cut,TCTP knock on reduction of A549 cells further confirmed the results from the opposite side.These results suggest that down-regulation of CSMD3 expression can lead to increased TCTP expression,which may further promote the function of A549 epithelial mesenchymal transformation of lung cancer cells by inhibiting the mir-200 s family expression,and further promote the migration and invasion of tumors.Part ?:PLOD2 affects the invasiveness and metastasis of the lung adenocarcinoma A549 cell line through CSMD3Objective: In the first part of the study,we found that the knockdown of CSMD3 can not only promote the proliferation of A549,but also lead to the up-regulation of PLOD2 gene expression.To further investigate how CSMD3 affects the function of A549 cells through PLOD2.To explore the effect of PLOD2 on the proliferation ability.Methods: In this study,tissue samples of 20 patients with lung adenocarcinoma were first collected to study the expression of PLOD2 gene in cancer-adjacent tissues,and the correlation between PLOD2 expression and survival of patients with lung adenocarcinoma was analyzed using the TCGA database.We then constructed the PLOD2 knockdown lentivirus expression vector and transfected the lung adenocarcinoma A549 cell line,and studied the effect of PLOD2 knockdown on the basic function of the A549 cell line.Results: 1.In the detection of 20 lung adenocarcinoma cancer-paracancerous tissue samples,it was found that the expression of PLOD2 gene in cancer tissue was significantly higher than that in paracancerous tissue(P<0.05).2.Analysis of the data of lung adenocarcinoma in the TCGA database showed that the expression of PLOD2 was significantly correlated with the survival period of patients with lung adenocarcinoma(P<0.01),in which the median survival period of the low expression group was 84.1 months,and the median survival period of the high expression group was only 52 months,that is,the survival period of patients with high expression of PLOD2 was short.3.In A549 lung adenocarcinoma cells that were artificially suppressed with PLOD2,PLOD2 knockout significantly increased migration efficienc(P<0.05),and in the transwell test,the number of cells passing through the transwell chamber was significantly reduced compared with the control group(P<0.05).Conclusions: It was found that the expression of PLOD2 in non-small cell lung cancer was higher than that in adjacent tissues,and the high expression of PLOD2 was closely related to the poor prognosis of patients with lung adenocarcinoma.The first part of the study found that CSMD3 knockdown can lead to increased expression of PLOD2.However,the effect of PLOD2 knockdown on the cell line A549 of lung adenocarcinoma was more complex,which showed that PLOD2 knockdown could inhibit the proliferation and invasion of the cell line A549 of lung adenocarcinoma,but the migration ability was improved.