| Background and objectiveLung cancer is the world’s main cancer-related death disease.It is one of the malignant tumors that cause high mortality in patients.It seriously harms human health and life.Lung cancer can be divided into two types:small cell lung cancer and non-small cell lung cancer.Lung cancer,80%to 85%of lung cancers belong to non-small cell lung cancer(NSCLC).Lung adenocarcinoma is a common subtype of non-small cell lung cancer,accounting for about 40%of lung cancers.Because there are no obvious symptoms in the early stage of onset,70%to 80%of lung cancer patients are already in the middle and advanced stages at the time of diagnosis,and the 5-year survival rate is less than 20%.With the continuous development and progress of clinical medicine,in addition to conventional surgery,chemotherapy and chemotherapy,the development of targeted therapy,immunotherapy and other new broad-spectrum anti-cancer drugs has also made great progress in the treatment of lung adenocarcinoma.Targeted therapy has developed rapidly since its birth and has greatly improved the prognosis of NSCLC patients with positive driver genes.Even so,drug resistance and high metastasis rates are the main causes of treatment failure and death.Therefore,finding more driving genes that may participate in the occurrence and development of lung adenocarcinoma and researching and developing high-efficiency and low-toxicity targeted drugs are the top priority for basic and clinical research on lung cancer.The DIAPH3 gene is located in the long arm of human autosomal 13 long arm 2,zone 1,and its product belongs to the Formin protein family.This protein can participate in physiological activities such as actin remodeling,cell morphology changes,movement and adhesion regulation.A number of studies have shown that the expression of DIAPH3 gene is related to auditory nervous system diseases and silicosis,and is related to the occurrence and metastasis of breast cancer,hepatocellular carcinoma,prostate cancer,pancreatic cancer and other tumors.Guo et al.found that DIAPH3 is an up-regulated gene in lung adenocarcinoma and promotes the growth of lung adenocarcinoma cells.There are few studies on the occurrence and development mechanism of DIAPH3 in lung adenocarcinoma.Through the expression of DIAPH3 in the cancer genome atlas(TCGA)database and the analysis of the clinical information and survival information of lung adenocarcinoma patients by immunohistochemistry,To study the relationship between the DIAPH3 gene and the prognosis of patients with lung adenocarcinoma,and to study the effect of DIAPH3 on the proliferation and migration of lung adenocarcinoma cells and the mechanism that may promote the development of lung adenocarcinoma through in vitro cell experiments,in order to judge the prognosis of the tumor and clinical treatment The formulation of the plan plays a certain reference role.Methods1.Obtain the influence of different DIAPH3 mRNA expression levels on the survival of lung adenocarcinoma and lung squamous cell carcinoma through the GEPIA database.2.Obtain the expression data of DIAPH3 mRNA in lung adenocarcinoma and related pathological parameters through The Cancer Genome Atlas(TCGA)database,and incorporate all data into the study to analyze the relationship between its expression level and the clinicopathological characteristics of lung adenocarcinoma patients.The survival data obtained were included in the study to analyze the relationship between DIAPH3 mRNA expression,clinicopathological characteristics and overall survival(OS).3.Collected 55 cases of lung adenocarcinoma patients diagnosed by pathology from January to December 2015 in the Department of Thoracic Surgery,First Affiliated Hospital of Zhengzhou University,of which 26 cases collected corresponding paracancerous tissue specimens.Immunohistochemistry was used to detect the expression of DIAPH3 protein and analyze its relationship with tumor size,lymph node metastasis,distant metastasis,and OS in patients with lung adenocarcinoma.4.The lung adenocarcinoma cell lines A549 and HCC827 with high expression of DIAPH3 mRNA were screened by PCR.5.The expression of DIAPH3 gene in lung adenocarcinoma cell lines A549 and HCC827 was knocked down by plasmid transfection,and the overexpression and knockdown efficiency were verified by real-time fluorescent quantitative PCR and Western Blot(WB)methods.6.CCK-8 and plate cloning experiments were used to test the effect of DIAPH3 on cell proliferation;cell scratch test and Transwell migration test the effect of DIAPH3 on cell migration,7.Flow cytometry was used to detect the effect of DIAPH3 expression on apoptosis and cell cycle.8.WB was used to detect the effect of DIAPH3 expression on the expression of E-cadherin and vimentin,which are markers of epithelial-mesenchymal transition(EMT).9.Scratch,Transwell experiment to detect the effect of adding Wnt/β-catenin signaling pathway inhibitor XAV939 on cell migration.10.The lung adenocarcinoma cell line with high expression of DIAPH3 was added to the Wnt/β-catenin signaling pathway inhibitor XAV939,and WB was used to detect the effect of the inhibitor on the expression of E-cadherin and vimentin,the markers of EMT.Results1.GEPIA database analysis showed that high expression of DIAPH3 mRNA is a poor prognostic factor for patients with lung adenocarcinoma,and the expression level of DIAPH3 mRNA has nothing to do with the survival of patients with lung squamous cell carcinoma.2.DIAPH3 mRNA is highly expressed in lung adenocarcinoma and is related to lymph node metastasis,TNM staging and survival of tumor patients(all p<0.05).Multivariate analysis showed that high expression of DIAPH3 mRNA,N1-3 and clinical stages Ⅲ-Ⅳ were the influencing factors of patients’ OS(P<0.05).3.The expression level of DIAPH3 protein in cancer tissues was significantly higher than that in adjacent tissues(p<0.05),and was significantly correlated with lymph node metastasis and overall survival(p<0.05).COX analysis showed that the expression of DIAPH3 protein and lymph node metastasis were risk factors for OS(p<0.05).4.Cell experiments show that overexpression of DIAPH3 can promote cell proliferation,migration and transformation to EMT;reducing DIAPH3 expression can inhibit cell proliferation,migration and EMT transformation,block cell cycle in G0/G1 phase,and promote cell apoptosis(p<0.05).5.After adding the inhibitor XAV939,it inhibits the migration of lung adenocarcinoma cells.6.The expression of E-cadherin protein increased,and the expression of E-cadherin protein increased with the increase of the concentration of XAV939.The expression of vimentin protein decreased,and the expression of vimentin protein decreased with the increase of the concentration of XAV939.DIAPH3 may promote the transformation of tumor cells to EMT through Wnt/β-catenin.ConclusionDIAPH3 is highly expressed in lung adenocarcinoma,and may promote proliferation and migration of lung adenocarcinoma through Wnt/β-catenin signaling pathway. |
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