The Expression And Clinical Significance Of TRIM37 In Non-small Cell Lung Cancer

Background:Lung cancer is the most common cancer in the world.It is also the main cause of cancer death.Every year,there are 1.6 million new cases and 1.38 million deaths.Lung cancer can be divided into non-small cell lung cancer(NSCLC)and small cell lung cancer(SCLC).NSCLC accounts for 80-85%of all lung cancer.Although more and more treatments such as surgery,chemotherapy,radiotherapy and targeted therapy have been applied in clinic,the 5-year overall survival rate of NSCLC is still less than 20%An important reason for the high mortality of NSCLC is the lack of effective early diagnosis methods.The clinical manifestation and prognosis of NSCLC mainly depend on its stage,and the early treatment effect is more significant.However,due to the lack of effective early diagnosis methods,NSCLC is often diagnosed only when local or distant metastasis occurs in the late stage,so there are still many difficulties in improving the survival rate of non-small cell lung cancer.Studying new prognostic factors as biomarkers of NSCLC may be an important driving force for the advancement of early diagnosis technology of NSCLC.Previous studies have shown that TRIM37 expression is increased in hepatocellular carcinoma,pancreatic cancer,colon cancer,breast cancer and other tumors,and is associated with the prognosis of tumors,but the role of TRIM37 in lung cancer is still unclear.In this study,we downloaded relevant data from GEO and TCGA databases and analyzed the expression level of TRIM37 in NSCLC patients and adjacent tissues,as well as its correlation with prognosis.The effect of interfering and overexpressing TRIM37 on cell proliferation,migration and invasion was observed in lung cancer cells.The relationship between Akt signal and TRIM37 was further analyzed by using Akt inhibitor to clarify the function of TRIM37 in lung cancer and the corresponding signaling pathway.Part Ⅰ:The expression of TRIM37 in lung cancer and its correlation with prognosisObjective:To analyze the expression of TRIM37 in cancer and adjacent tissues of NSCLC patients and its correlation with prognosis.Methods:The gene expression and clinical information of 1145 NSCLC patients were downloaded from TCGA database(https://cancergenome.nih.gov/).The patients were divided into high-expression and low-expression TRIM37 groups by X-tile method.Survival analysis was performed by Kaplan-Meier method and log-rank test.Seven groups of data(GSE27262,GSE30219,GSE31210,GSE33532,GSE30219,GSE7670 and GSE10072)were collected from the GEO database to analyze the correlation between TRIM37 expression level and clinicopathological features,as well as its value in the diagnosis of lung cancer.KM-Plotter(http://www.kmplot.com/analysis/index.php?P=service&cancer=lung)was used to analyze the relationship between the expression of TRIM37 and the survival time of NSCLC patients.95 specimens of lung adenocarcinoma and corresponding paracancerous tissues From January 2012 to January 2013 were collected;30 specimens of lung adenocarcinoma and corresponding paracancerous tissues collected from August 2016 to March 2017 were frozen in liquid nitrogen for RNA extraction,all samples were enrolled from the First Affiliated Hospital of Suzhou UniversityThe expression of TRIM37 in cancer and adjacent tissues was detected by fluorescence quantitative PCR(30 cases by frozen)and immunohistochemistry(95 cases by fixed).The correlation between the expression of TRIM37 and survival was analyzed by Kaplan-Meier method and log-rank test in patients were enrolled in 2012-2013.Results:Analysis of TCGA data showed that the expression of TRIM37 in lung adenocarcinoma(ADC)and squamous cell carcinoma(SSC)was significantly higher than that in adjacent tissues.Survival analysis showed that the high expression of TRIM37 in ADC was significantly correlated with the decrease of survival time,but the expression of TRIM37 in SSC had no significant effect on survival time.The results of KM-Potter analysis showed that there was no significant difference in survival time between the high and low expression groups of TRIM37 in all NSCLC patients.There was no significant difference in survival time between the high and low expression groups of TRIM37 in SCC patients,but the survival time of the patients with high expression of TRIM37 in ADC was significantly reduced.There was no significant difference in survival time between high and low expression groups of TRIM37 in smoking NSCLC patients,while the survival time of non-smoking NSCLC patients with high expression of TRIM37 was significantly reduced.Analysis of GEO data showed that the increased expression of TRIM37 was significantly correlated with TNM stage and tumor size in ADC patients.Univariate and multivariate analysis showed that TRIM37 might be an independent prognostic factor in ADC patients.ROC analysis showed that TRIM37 had significant diagnostic significance.The proportion of men and women in 95 ADC patients was basically balanced.Stage Ⅰ-Ⅱaccounted for more than 60%of the patients and 55%of the non-smokers.The results of fluorescence quantitative PCR and immunohistochemistry showed that the expression level of TRIM37 in cancer tissues was significantly higher than that in adjacent tissues,and the increase of TRIM37 expression was related to the decrease of survival timeConclusions:The expression level of TRIM37 in cancer tissues was significantly higher than that in adjacent tissues.The high expression of TRIM37 was significantly correlated with the decrease of survival time in lung cancer patients.The increased expression of TRIM37 is associated with TNM stage and tumor size in ADC patients,and is also an independent prognostic factor for ADC patients.TRIM37 has high specificity and sensitivity as a diagnostic marker of ADC,and may be a potential prognostic and diagnostic marker for ADC patients.Part Ⅱ.TRIM37 targets AKT in the growth of lung cancer cellsObjective:To analyze the role of TRIM37 in lung cancer cells and its possible molecular mechanism.Methods:The expression levels of TRIM37 in lung cancer tissues,adjacent tissues,lung cancer cell lines H292,H358,H1299,A549,11975 and human bronchial epithelial cells 16HBE were analyzed by fluorescence quantitative PCR,Western blot or immunohistochemistry.To construct lentiviruses interfering with and overexpressing TRIM37,infect different lung cancer cells and change the expression level of TRIM37 CCK-8 kit was used to detect the effect of TRIM37 expression on proliferation of lung cancer cells.Annexin V-FTTC/PI staining was used to observe the effect of TRIM37 expression on apoptosis of lung cancer cells.TRANSWELL method was used to analyze the effect of RIM37 expression on invasion and metastasis of lung cancer cells.The effects of TRIM37 expression on the expression of Bcl2,Bax,Akt and p-Akt in lung cancer cells were analyzed by fluorescence quantitative PCR and Western blot.Then PI3K/Akt inhibitor LY294002 was used to treat the cells.The relationship between Akt signal and TRIM37 was further analyzed to clarify the function of TRIM37 in lung cancer and the corresponding signaling pathway.Then we inoculated H358 cells from control group and TRIM37 interference group into nude mice to observe the effect of TRIM37 on the proliferation of lung cancer cells in vivoResults:The results of quantitative fluorescence PCR and Western blot showed that the expression levels of TRIM37 in cancer tissues were significantly increased,and the expression levels of TRIM37 in cancer tissues were also up-regulated.Compared with 16HBE cells,the expression of TRIM37 was significantly increased in lung cancer cell lines,especially in H292 and H358 cells.Compared with other lung cancer cell lines,the expression of TRIM37 in H1299 cells was relatively low.Therefore,H292,H358 and H1299 cells were selected for subsequent experimental study.In H292 and H358 cells,the proliferation,invasion and migration ability of cells at 24,48 and 72 hours after interfering with TRIM37 expression were significantly lower than those in control group,and the apoptotic level was significantly higher than that in control group.The expression of Bcl-2 and BAX in TRIM37-down-regulated cells was significantly decreased,while the expression of BAX was significantly increased.There was no significant difference in Akt level between control cells and TRIM37-interfered cells,but the level of p-Akt protein in TRIM37-interfered cells decreased significantly.Overexpression of TRIM37 in H1299 cells can promote cell proliferation,invasion and metastasis,but the ability of cell proliferation,invasion and metastasis decreased significantly after PI3K/Akt inhibitor LY294002 was used.Overexpression of TRIM37 can up-regulate the expression of p-Akt and Bcl2 and inhibit the expression of Bax,while PI3K/Akt inhibitor LY294002 can inhibit the expression of p-Akt and Bcl2 and promote the expression of Bax.The results of in vivo experiments showed that compared with the control group,the proliferation ability,volume and weight of tumors in TRIM37 interference group decreased significantlyConclusions:TRIM37 can regulate the expression of Bcl2,inhibit the expression of Bax and promote the proliferation,invasion and metastasis of lung cancer cells by activating Akt signaling pathway.