Study On The Correlation Between UBE2S Gene And Lung Adenocarcinoma Based On TCGA And Its Functional Mechanism In Lung Adenocarcinoma

Chapter 1 To analyze the expression and correlation of UBE2S gene in lung adenocarcinoma based on TCGABackground:Adenocarcinoma of Lung,the most common type of lung cancer,is classified as non-small cell lung cancer(NSCLC)along with lung squamous cell carcinoma and large cell lung cancer.Epidemiological studies have found that patients with adenocarcinoma of the lung account for up to 62%of patients with lung cancer and have no smoking history.Among lung cancer patients without smoking history,the incidence of lung adenocarcinoma was 3.4 times as that of lung squamous cell carcinoma.On the other hand,the incidence possibility of Adenocarcinoma bronchoalveolar subtype in non-smoking women are 2-4 times as that in men,and patients with lung adenocarcinomas account for 41.4%of female patients with lung cancers.75%of female twins with lung cancer are Monozygotic twins,indicating that Lung cancer may be closely associated with familial inheritance and abnormal gene expression.In recent years,the establishment of TCGA has provided a rare opportunity to explore the molecular mechanism of lung adenocarcinoma.Objective:1.In this study,the abnormal expressed genes in TCGA lung adenocarcinoma sample were screened,and the possible clinical application value of the abnormal expression of this target gene in lung adenocarcinoma was proposed through bioinformatics analysis.2.With filtering these clinical samples,the correlation between abnormal expression of selected genes and the clinicopathological characteristics of lung adenocarcinoma was verified,and the clinical application value and research value of this gene were determined.Methods:1.Firstly,57 cases of lung adenocarcinoma samples,which had both tumor tissue and para-carcinoma tissue related data as well as the pathological information,were downloaded from the TCGA website.Then analyzed and filtered the genome data of these 57 sample,and selected the transcription of gene with highest expression,used the TMM(Trimmed Mean of M-values)algorithm for standardization of the filtered data.Then,the list of differentially expressed genes was obtained through analyzing the discrete value,linear value and Log2 value,and combined with literature screening.The differentially expressed genes in the list were randomly condensed to determine the target genes.Bioinfomatics analysis was conducted on the expression level of the target gene and the clinical sample data of the corresponding 522 cases from TCGA lung adenocarcinoma data,then analyzed the correlation between the target gene and the clinical data of lung adenocarcinoma.2.Immunohistochemical was used to detect the expression of antibody against the target gene in tumor tissues and para-carcinoma tissue of 30 clinical samples of lung adenocarcinoma,and combined with the bioinformatics analysis of the clinical data of these samples to verify the correlation between abnormal expression of target gene and pathological staging of lung adenocarcinoma,aiming to determine its clinical application valueResults:1.With screening the differentially expressed genes in lung adenocarcinoma samples in TCGA combined with literature screening,it was confirmed that the abnormal expression of UBE2S in lung adenocarcinoma had potential research value Bioinformatics analysis of the clinical information of lung adenocarcinoma samples in TCGA and expression level of UBE2S showed that the expression of UBE2S in lung adenocarcinoma tumor tissues and para-carcinoma tissues was significantly different(P<0.05),Difference analysis showed that the expression of UBE2S in lymph node metastasis(N metastasis)and T stages(P<0.05)were significantly different.Correlation analysis showed that there was a significant and positive correlation between the expression of UBE2S in lymph node metastasis(N metastasis)and pathological stages(P<0.05).2.Bioinformatics analysis of the expression of UBE2S antibody in lung adenocarcinoma tumor tissues and adjacent tissues of 30 samples and clinical data showed that the expression of UBE2S antibody in lung adenocarcinoma tumor tissues and adjacent tissues was significantly different(P<0.05).Difference analysis showed that the expression of UBE2S antibody in each T stages showed significant difference(P<0.05).Correlation analysis showed that the expression of UBE2S antibody was significantly correlated with the T stages,and UBE2S expression was positively correlated with T stages(P<0.05).There was a significant difference in the expression of UBE2S in lung adenocarcinoma tumor tissues and adjacent tissues(P<0.05).Conclusion:In this study,the expression of UBE2S was found to be lower in the pre-stage of lung adenocarcinoma,and the expression level increased with the progression of lung adenocarcinoma,increase of tumor and lymphatic metastasis.The abnormal expression of UBE2S in lung adenocarcinoma may be a potential diagnostic indicator for detecting the clinical staging of lung adenocarcinoma.Chapter 2 The effects of UBE2S on the function of lung adenocarcinoma cellsBackground:Ubiquitin-coupled enzyme E2S(UBE2S)is a member of E2 proteins family in the process of ubiquitination.In normal tissues.UBE2S plays key roles in cell cycle regulation,cell differentiation,and DNA repair,so UBE2S is inevitably involved in the occurrence and development of tumors.Plenty of papers have demonstrated the abnormal expression of UBE2S occurred in breast cancer,oral squamous cell carcinoma,cervical cancer,hepatocellular carcinoma and renal carcinoma.However,the effects of UBE2S on the function of lung adenocarcinoma cells have not been explained.Objective:In this study,UBE2S gene was knockout or overexpressed in lung adenocarcinoma cancer cells through slow virus infection.and detected the effects of UBE2S knockout and overexpression on cell counting,cell apoptosis,clone formation cell vitality,etc.in lung adenocarcinoma cells.This study aimed to explicit the impacts of UBE2S knockout and overexpression on lung adenocarcinoma cancer cells,and explores the important roles of UBE2S in lung adenocarcinoma development.Methods:Established a model of lung adenocarcinoma cells with UBE2S knockout or overexpression through lentivirus infection.Then through detection of Celigo cell counting,FACS cell apoptosis rate detection,clonal formation.Caspase3/7 activity assay and MTT cell viability,the effects of UBE2S knockout or overexpression on cells indicators such as lung adenocarcinoma cell counting,apoptosis,clonal formation and cell viability was observed.Results:1.Celigo cell counting detection found that compared with the negative gene knockout lung adenocarcinoma cancer cells,lung adenocarcinoma cancer cell quantity and quantitative variation with time are relatively slower when UBE2S was knockout,and had significant difference(P<0.05).Moreover,FACS detection showed that cell apoptosis rate increased significantly(P<0.05).BrdU DNA synthesis detection found that DNA synthesis decreased significantly(P<0.05).Clone formation test found that cloning formation decreased significantly(P<0.05).Caspase-3/7 test showed that caspase-3/7 activity increased significantly(P<0.05).MTT cell vitality test found that the absorption rate at 490 nm and absorption rate changes with time are much slowly and had significant differences(P<0.05).2.In the control experiment of overexpression of UBE2S gene of NCI-H1975,NCI-H1299,and 95-D cell lentivirus infection and control group,compared with negative overexpression lung adenocarcinoma cells,the apoptosis rate of FACS cells was decreased,the colony formation assay was increased,and the activity of caspase3/7 was decreased in NCI-H1299 cell overexpression group,with significant differences(P<0.05).Conclusion:1.the deletion expression of UBE2S gene in lung adenocarcinoma cells could inhibit cell proliferation and clone formation,promote apoptosis and reduce cell viability.UBE2S gene might be a new target in the treatment of lung adenocarcinoma.2.Overexpression of UBE2S gene in NCI-H1299 cells could decrease the apoptosis rate and caspase3/7 activity,and increase colony formation.The overexpression of UBE2S in lung adenocarcinoma cells may be a factor contributing to the deterioration of lung adenocarcinoma and may be a potential indicator for judging the degree of lung adenocarcinoma deterioration.Chapter 3 Mechanism of UBE2S as a Tumor-Driven Gene in Lung AdenocarcinomaBackground:Previous studies have shown that the loss of UBE2S gene expression in lung adenocarcinoma cells can inhibit cell proliferation and cloning,promote,cell apoptosis and reduce cell viability.In this paper,we study the downstream genes and clarify their mechanism as driver geneObjective:1.In this study,the whole gene expression profile microarray technology was used to detect the effects of UBE2S knockout on the whole-gene expression in lung adenocarcinoma cells,and to screen its downstream gene.2.The molecular mechanism of UBE2S was verified by detecting the expression of these downstream genes at the mRNA levelMethods:1.Detected the gene expression in UBE2S knockout and negative lung adenocarcinoma cells in the control grop through the whole gene expression profile microarray technology,with IPA(Ingenuity Pathway Analysis)to analyze the differences genes in classical pathway enrichment,upstream regulatory factors,disease and function,control effects,intermolecular interaction network,and screened the intense activation or inhibition target molecules upstream or downstream of UBE2S2.Western blot was used to analyze P53,NUPR1 and ILK proteins in A549 cells of control group and UBE2S knockout group.3.By detecting and comparing the mRNA expression levels of the downstream target genes which is intensely regulated by UBE2S in lung adenocarcinoma cells,the molecular functional mechanism of UBE2S in lung adenocarcinoma cells was verified and provedResults:1.The gene expression of lung adenocarcinoma cells was changed by the knockout of UBE2S gene,and a total of 458 genes were significantly regulated(P<0.05),of which 171 genes were up-regulated and 287 genes were down-regulated.The classical pathways enrichment analysis of differentially expressed genes showed that the knockout of UBE2S gene activated the p53 signaling pathway,the integrin signaling pathway and the ILK signaling pathway,and inhibited the coagulation system signaling pathway,ATM signaling pathway and the acute response signaling pathway.Upstream regulatory factor analysis showed that after UBE2S gene knockout,NUPR1 gene was strongly activated,and there were 34 uniformly activated genes regulated by NUPR1.By analyzing the interaction between the NUPR1 gene and its downstream molecules,it was found that the NUPR1 gene plays a mediating role between UBE2S and upstream and downstream molecules2.The expression of P53 and NUPR1 was up-regulated after UBE2S knockout in A549 cells,while the expression of ILK was down-regulated by UBE2S knockout.3.It was found that low expression of UBE2S can lead to the intense down-regulation of ACSS1,NUPR1 PPP1R15A,SPAG5 and the intense up-regulation of CITED2.GADD45A.IGF2BP3Conclusion:1.The molecular mechanism of UBE2S gene deletion leading to the slow proliferation and increased apoptosis of lung adenocarcinoma cells may be influenced by the activation of p53 signaling pathway,integrin signaling pathway,ILK signaling pathway and other cancer inhibitory signaling pathways2.The low expression of UBE2S may reduce the degradation of the transcriptional regulator NUPR1 by affecting the ubiquitination/proteasome system,thereby inhibiting or mediating the cell cycle of lung adenocarcinoma cells and promoting their apoptosis.3.The low expression of UBE2S may activate CITED1,GADD45A and DDIT3 through NUPR1,and inhibit ACSS1,resulting in the activation of stress response,slowing cell cycle,and ultimately the apoptosis of lung adenocarcinoma cells.