| BackgroundLung cancer is one of the leading causes of cancer deaths worldwide,with an estimated 770,000 people will die from lung cancer in China in 2022.The 5-year survival rate of lung cancer patients is only 15-20%,which is attributed to diagnosis limitations and current clinical drug resistance.Non-small cell lung cancer(NSCLC)is a malignant tumor originating in the epithelium of the bronchial mucosa or alveolar epithelium,which is the most common type of lung cancer.In recent years,with the development of medical technology,new treatment methods including targeted therapy and immunotherapy have brought unprecedented survival benefits to some patients,but the overall cure rate and survival rate of NSCLC are still very low,especially for advanced patients.Therefore,it is urgent to explore novel drugs or combination therapy to improve the prognosis of patients with NSCLC.Epigenetic modification is a process of regulating gene expression without changing the DNA sequence.More and more evidence has suggested that epigenetic modification enzyme dysregulation is associated with the occurrence and development of cancer,targeting epigenome-related molecules is able to improve cancer symptoms.SETD1A(SET Domain containing 1A)is an epigenetic modifying enzyme,is a histone lysine methyltransferase containing SET domain,which is mainly expressed in the nucleus and distributed in many tissues of the body.It not only participates in histone modification,but also plays an important regulatory role in DNA damage repair,cell cycle,gene transcription,genomic imprinting and tumor development.H3K4me3(Histone 3 Lysine 4 trimethylation)is the trimethylation of the fourth lysine of histone 3,which is enriched on promoters near the transcription start site and acts as a histone code to identify gene promoters.It activates oncogene transcription through covalent modification and chromatin remodeling,and participates the development and prognosis of various malignant tumors.Studies have confirmed that H3K4me3 levels were elevated in lung adenocarcinoma cell lines and involved in abnormal transcriptional regulation,but the mechanism was not clear.SETD1A binds to histone H3K4 modification sites through the SET domain,and can catalyze histone H3K4 site trimethylation(H3K4me3)specifically.H3K4me3 is associated with the promoter region and early transcription region of activated genes,and can release transcriptional elongation,resulting in gene transcription level changes.SETD1A increases the expression of H3K4me3 modification level and induces gene activation,and participates in the occurrence and development of various malignant tumors.Studies have found that the expression of SETD1A is significantly increased in lung cancer,which can promote the proliferation of lung cancer cells and is related to the survival and prognosis of lung cancer.However,the biological role and mechanism of SETD1A in NSCLC remains largely unclear.In this study,the expression of SETD1A in NSCLC tissues and its relationship with clinical prognosis were clarified by bioinformatics analysis and clinical lung adenocarcinoma tissues.The effects of SETD1A differential expression on the proliferation,invasion and migration of NSCLC cells were studied through in vitro and in vivo experiments,and the molecular mechanism of SETD1A involved in the progress of NSCLC was preliminarily discussed.This study elucidated the role and mechanism of SETD1A in NSCLC,which may provide a new molecular target for the treatment of NSCLC.ObjectiveThrough the analysis of bioinformatics and clinical NSCLC tissues,the expression of SETD1A in NSCLC tissues and its relationship with clinical prognosis were determined.The effects of SETD1A differential expression on the proliferation,invasion and metastasis of NSCLC cells and the possible molecular mechanism were discussed through in vitro and in vivo experiments.Method1.Bioinformatics and clinical NSCLC tissue analysis:(1)The expression of SETD1A mRNA in NSCLC tissues and paracancerous tissues was analysed by utilizing TCGA database,and its relationship with survival and clinical TNM stage of lung cancer patients was analysed;(2)The expression of histone methylase in lung cancer and paracancerous tissues was searched by GEO database;(3)Clinical lung adenocarcinoma tissues samples were collected,and the expression of SETD1A protein in lung adenocarcinoma tissues and adjacent tissues was detected by immunohistochemical staining,and the correlation between SETD1A protein and clinicopathological parameters of patients was analyzed.2.In vitro experiments:(1)A549 and H1299 cells with relatively high expression of SETD1A were screened out by RT-qPCR for functional analysis;(2)The SETD1A knockdown A549/H1299 stably transformed cell line was constructed with SETD1A shRNA packaged with lentivirus,and the knockdown efficiency was verified by Western blot;(3)The cells were counted by trypan blue staining to observe the effect of SETD1A on the proliferation of NSCLC cells.and the effect of SETD1A on the expression of cell proliferation-related proteins was detected by Western blot assay;(4)The effects of SETD1A on the migration and invasion of lung cancer cells were detected by cell scratch assay and Transwell chamber assay;(5)The effects of SETD1A on the expression of EMT-related markers in A549 and H1299 cells were detected by Western blot assay;(6)Immunofluorescence assay was performed to detect the effect of SETD1A on the global H3K4me3 in A549 cells.CHIP assay was performed to detect the effect of SETD1A on H3K4me3 methylation modification of specific oncogene promoter region.Western-blot was performed to detect the protein level of proto-oncogene that was altered by transcriptional regulation.3.In vivo experiments:(1)Lentivirus-transfected A549/H1299 cells were subcutaneously injected into the back of nude mice,and the xenograft tumor model in nude mice was successfully established;(2)The effects of SETD1A on the size and weight of xenograft tumor in nude mice were measured;(3)The effects of SETD1A on EMT-related markers in nude mouse transplanted tumor was detected by RT-qPCR;(4)The effect of SETD1A on tumor oncogene transplantation in nude mice was detected by RT-qPCR.Result1.Bioinformatics and clinical NSCLC patient tissue analysis:(1)TCGA database analysis showed that SETD1A was frequently amplified and mutated in NSCLC,Compared with partial deletion or diploid,the amplification and mutation of SETD1A gene locus upregulated SETD1A transcription level in NSCLC.(2)Compared with the adjacent tissues,SETD1A mRNA expression was significantly increased in lung adenocarcinoma tissues,and the later TNM stage was,the higher the expression level of SETD1A mRNA was,indicating that SETD1A was positively correlated with the clinical stage of lung adenocarcinoma.(3)Kaplan-Meier survival curve analysis showed that SETD1A mRNA level is highly negatively correlated with the survival period of lung cancer patients,In addition,the mRNA levels of SETD1A methylation-related proteins(WDR5 and HCFC1)level were also negatively correlated with overall survival status,these results suggest that SETD1A is associated with poor prognosis in NSCLC patients.(4)The mRNA level of KMT2 family members(except SETD1A)has no significant difference in lung cancer tissues and adjacent tissues,indicating that SETD1A plays a unique role of KMT histone methyltransferase in lung cancer and may play a major role in the regulation of H3K4me3 in the progression of lung cancer.(5)SETD1A protein is highly expressed in lung adenocarcinoma tissues,mainly located in the nucleus,and is related to the clinical TNM staging.2.In vitro experiments:(1)Western blot showed that knockdown of SETD1A gene inhibited the expression of SETD1A and Non-P-CTNNB1 protein in NSCLC.(2)Compared with the control group,knockdown of SETD1A in A549 and H1299 could inhibit cell proliferation rapidly and significantly.(3)Knockdown of SETD1A could decrease the expression of A549 and H1299 cell proliferation-related proteins,but the protein expression of ERK signal(PMAPK1)was not down-regulated significantly.(4)Wound healing assay results showed that knockdown of SETD1A gene in A549 cells inhibited the migration ability of NSCLC cells.Transwell assay also showed that knockdown of SETD1A gene in A549 and H1299 cells inhibited the migration and invasion ability of A549 and H1299 cells.(5)Knockdown of SETD1A gene up-regulated the expression of epithelial cell marker CDH1 protein in A549 and H1299 cells,and down-regulated the expression of mesenchymal cell marker(CDH2,VIM,ZEB1 and SNAIL1)protein.(6)The effect of SETD1A knockdown in A549 cells was obvious,but the SETD1A knockdown did not lead to a significant reduction in global H3K4me3 signal.However CHIP assay found that the modification of H3K4me3 in the transcriptional regulatory region of the proto-oncogene decreased correspondingly,indicating that the transcription level of the proto-oncogene was inhibited to a certain extent.Western blot assay found that the protein expression level of proto-oncogene was also significantly down-regulated.3.In vivo experiments:(1)The xenograft model of NSCLC in nude mice was successfully constructed.Compared with the control group,downregulation of SETD1A expression significantly inhibited the growth rate of NSCLC xenograft in nude mice.(2)Knockdown of SETD1A gene significantly up-regulated the expression of epithelial marker mRNA and down-regulated the expression of mesenchymal marker mRNA in NSCLC xenograft in nude mice.(3)Knockdown of SETD1A gene can reduce the expression of epigenetic modification enzymes and proto-oncogenes in NSCLC xenograft in nude mice.Conclusion1.SETD1A is involved in the development of NSCLC and is associated with poor clinical prognosis of patients.2.SETD1A regulates the EMT process and promotes the invasion and migration of NSCLC cells.3.SETD1A may regulate various proto-oncogene of NSCLC through H3K4me3 levels,and further promote the progression of NSCLC. |
PDF Full Text Request