| Background:Lung cancer is one of the most common malignant tumors in humans with high mortality and morbidity.Among them,non-small cell lung cancer(NSCLC)is the main type of lung cancer,accounting for 80-85%of lung cancer.According to cell type,NSCLC is mainly divided into Lung squamous cell carcinoma(LUSC)and lung adenocarcinoma(LUAD).Tumor progression is closely related to many basic biological processes,such as post-translational modification,and ubiquitination is one of the most important post-translational modifications.The ubiquitin system plays an important role in regulating the massive degradation of proteins,involving processes such as cell cycle regulation,signal transduction,receptor regulation,and apoptosis.The ubiquitination process is mediated by E1 ubiquitin-activating enzymes(E3s),E2 ubiquitin-conjugating enzyme(E2s)and E3 ubiquitin ligases(E3s).E1s activates ubiquitin(Ub)and transfers it to E2s to bind to the C-terminus of Ub with a thioester bond,and then E3 regulates the final step of mediating Ub transfer by interacting with the E2-Ub complex and a specific substrate.The entire ubiquitination process plays an important role in tumor development by regulating the degradation of oncogene products and tumor suppressor factors.E3s contains three families,including the E6-AP COOH terminus(HECT)family,the RING finger-containing family,and the RING-Between RING-RING.Family,RBR)family.In mammals,the tripartite motif-containing(TRIM)contains more than 80 members,most of which are E3 ubiquitin ligases.Although not all members of the TRIM family have been identified as E3 ubiquitin ligase activity,and only a few have been functionally validated,the TRIM gene family has attracted much attention in recent years and has become the focus of E3s research.According to previous studies,TRIM family genes are closely related to the occurrence and development of malignant tumors.The relationship between them can be roughly divided into four categories,including carcinogenic regulation,such as TRIM24,TRIM25,TRIM27,TRIM28,anti-carcinogenic regulation,such as TRIM3,TRIM8,TRIM13,TRIM16,the regulation of tumor metastasis,such as TRIM8,TRIM11,TRIM16,TRIM62,and the regulation of DNA repair,such as TRIM29.The same TRIM family member may play different roles in different tumors.In the study of animal models of liver cancer mice,experiments have shown that TRIM24 can inhibit the occurrence and development of tumors.Another member of the TRIM family,TRIM29,has been studied in a variety of tumors,including gastric cancer,bladder cancer,esophageal cancer,and pancreatic cancer.Studies have shown that up-regulated TREM29 expression is associated with poor prognosis.Another study reported that TRIM29 inhibits the epithelial-mesenchymal transition(EMT)process by inhibiting the TWIST gene.In conclusion,a comprehensive study of TRIM family protein function,and the relationship between tumors is very important.TRIM family proteins play an important role in tumor progression through their promotion and inhibition in different cancers However,due to the large number of members and complex mechanisms,the relationship between different TRIM proteins and tumor tissues and the molecular mechanism are not fully understood.According to current literature studies,research on the TRIM family and lung cancer has been very limited to date.In order to better understand the influence of TRIM family members and the prognostic value of lung cancer,this study used the Cancer Genome Atlas(TCGA)database to download clinical data and RNA sequence data of LUSC and LUAD to analyze the genes of the entire TRIM family members.The relationship between expression and prognosis,screening for target factors in the TRIM family,and further analysis of the biological effects of the target on lung cancer cells at the cellular level.This study analyzed the relationship between TRIM family proteins and lung cancer.Screening of target factors is of great significance for drug research and clinical diagnosis of targeted targeted therapy for lung cancer.PartⅠ:Relationship between differential expression of TRIM gene family and clinical prognosis in non-small cell lung cancerObjective:To analyze the relationship between TRIM gene family differential expression and clinical prognosis in non-small cell lung cancer(NSCLC),and to screen target factors in the TRIM gene family for clinical diagnosis or prognosis of non-small cell lung cancer.Method:1.Use the Tumor Genome Atlas(TCGA)dataset to collect TRIM family member RNA sequence data and clinical data for lung cancers(LUSC)and lung adenocarcinoma(LUAD),the main cancer types of non-small cell lung cancer,by DE language Eseq pair RNA-seq data was filtered and normalized,and Heml(Heatmap Illustrator,version 1.0)software senerated heat maps showing differential expression of TRIM family members in LUSC and LUAD samples.Integrate analysis of clinical data and RNA sequence data between LUSC and LUAD patients,combining two cancer types to select members with differences in expression in the TRIM family.2.Online Kaplan-Meier-plotter and univariate Cox proportional hazard model were used to analyze the correlation between all TRIM gene family members and clinical prognosis,and to screen TRIM family members with significant prognostic value.3.The expression of TRIM15 was analyzed using a series of matrix fues from the GSE75037,GSE19804 and GSE43458 databases in the Gene Expression Omnibus(GEO)database.4.Using R2 genome analysis and visualization platform 36742 at dataset,the difference of TRIM 15 expression between normal tissue and tumor tissue was analyzed.K-M survival curve was drawn in the dataset(36742 at),and the relationship between TRIM 15 expression and prognosis was analyzed by univariate COX regression.5.Use the cancer genomics database cBioPortal to download EGFR,LKB1 and KRAS wild-type and mutant LUAD patient data,and p53 wild-type and mutant LUAD and LUSC patient data,analyze TRIM 15 expression differences,and plot KM survival curve to analyze TRIM 15 expression and prognosis.Result:1.The TRIM family has 19 members in LUSC and 14 members in LUAD,respectively,showing significant differential expression,and there are 10 members TRIM2,TRIM 15,TRIM16L,TRIM59,TRIM17,TRIM72,TRIM9,TRIM58,MEFV and TRIM45.Significant differential expression was shown in both LUSC and LUAD(log2FC change value≥1,adjusted p value<0.05).2.Kaplan-Meier survival analysis was used to map K-M survival curves for members with significant differential expression in LUSC and LUAD.The univariate Cox proportional hazards model was used to analyze the prognostic value of TRIM gene family members.The results showed that three members TRIM2,TRIM15 and TRIM58 showed significant prognostic value in both LUSC and LUAD.Among them,TRIM15 in lung squamous cell carcinoma(LUSC),TRIM15 Log2FC was 5.16(p =0.00575),and in lung adenocarcinoma,TRIM15 Log2FC was 6.37(p = 6.78E-07).Up-regulated TRIM15 was associated with poor prognosis in LUSC(HR 1.353;95%CI 1.023-1.789;p = 0.034)and LUAD(HR 1.560;95%CI 1.159-2.101;p = 0.003)and could be used as a potential target for analysis.3.The expression of TRTIM15 was analyzed using a series of matrix files from the GSE75037,GSE19804 and GSE43458 databases.The expression of TRIM15 in tumor tissues was significantly higher than that in normal tissues(p<0.0001 in GSE75037 and GSE43458,p=0.007 in GSE19804).4.R2 genome analysis and visualization platform plotting the results of the 36742_at dataset expression difference analysis revealed that TRIM15 was up-regulated in LUAD tissues(p =2e-03).At the same time,KM survival curves were plotted in the dataset(36742 at).Univariate COX regression showed that TRIM15 was significantly associated with the prognosis of LUSC and LUAD,respectively(p = 0.043,HR = 1.3(1.01-1.68)in LUSC,p=1.2e-06),HR= 1.81(1.42-2.31)in LUAD.5.The expression of TRIM15 in patients with EGFR mutations was significantly higher than that in wild-type patients.Among patients with KRAS-mutant LUAD,patients with low TRIM15 expression had a better prognosis than patients with high TRIM15 expression(p=0.044).Conclusion:Ten members of the TRIM family showed significant differential expression in both LUSC and LUAD.Three members of TRIM2,TRIM15 and TRIM58 showed significant prognostic value in both LUSC and LUAD,and TRIM 15 could be used as a potential target.Part Ⅱ:Expression and role of TRIM15 in non-small cell lung cancerObjective:To confirm the differential expression of the target factor TRIM 15 in the TRIM gene family in lung cancer patients,and to examine the effect of TRIM 15 on the biological function of lung cancer A549 cells,and further study the expression and role of TRIM 15 in non-small cell lung cancer.Method:1.Quantitative real-time PCR was used to analyze the expression of TRIM 15 in tumor tissues and paired normal tissues of 17 patients with primary NSCLC;immunohistochemistry(IHC)was used to confirm the presence of 35 pairs of NSCLC tumor tissues and paired normal tissues in tissue microarrays.Difference in expression of TRIM15;2.The expression of TRIM15 in human lung adenocarcinoma cell line A549 was knocked down by siRNA to construct SiTRIM 15-1 and SiTRIM 15-2 cell lines.MTT assay was used to detect the proliferation of lung cancer cells and SiTRIM15-1 and SiTRIM15-2 cells,cell migration was detected by cell scratching,and cell cycle was detected by flow cytometry.4.Use the gene set enrichment analysis(GSEA)to predict the biological process of TRIM 15 action.Result:1.Quantitative real-time PCR results showed that TRIM 15 was significantly higher in NSCLC tissues than in matched normal tissues(p = 0.0009)in 17 pairs of matched cancer tissues and normal tissues.Tissue microarray analysis showed that expression of TRIM 15 was significantly higher in tumor tissues in 35 pairs of tissues compared to matched normal tissues(p<0.0001).Analysis of immunohistochemical staining results showed that the higher the TRIM 15 expression,the greater the proportion of tumor tissue.2.Western blotting confirmed the successful construction of lung cancer cells knocking down TRIM15.3.The proliferation ability and cell migration ability of lung cancer cells knocking down TRIM15,SiTRIM15-1 and SiTRIM15-2,were significantly lower than those of control group with transfected empty vector.Compared with the control cells,the proportion of cells in the G0/G1 phase of siTRIM15-1 cells increased,and the number of cells in the G2/M phase decreased.4.Gene set enrichment analysis showed that high expression of TRIM15 is closely related to glycolysis,oxidative phosphorylation,cell cycle and biological processes related to proteasome and interferon(p values are less than 0.001).Conclusion:TRIM15 is highly expressed in tumor tissues of lung cancer patients.Knockdown of TRIM15 can inhibit the proliferation,migration and cycle of lung cancer cells A549.TRIM15 may be involved in cell glycolysis,oxidative phosphorylation,cell cycle,proteasome and interferon-a.Biological process. |
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