Role And Mechanism Of TRIM33 In Regulating MCM7 Function Through SIRT1-dependent Deacetylation In Lung Cancer

Background:Lung cancer is one of the most common malignant tumors and cancer with the highest mortality rate in the world,and non-small cell lung cancer(NSCLC)is the main type of lung cancer.Although the clinical treatment of lung cancer has made great progress in the past few decades,the current 5-year overall survival(OS)rate of lung cancer is only 18%-19%,and the OS of NSCLC is about 15%.The relatively low OS of lung cancer patients may be attributed to the lack of highly sensitive early screening diagnosis and precisely targeted therapy.Therefore,finding new molecular targets is of great significance for improving the survival rate and prognosis of lung cancer patients.TRIM33(Tripartite Motif Containing 33)is a member of the Tripartite Motif Containing 33(TRIM)family.Since members of this family generally have a RING domain,it is regarded as a typical type of E3 ubiquitin ligase,and is widely involved in immunity,inflammation,tumors and other physiological and pathological processes.Among them,the function of TRIM33 in tumors is relatively complex,and it may play different roles in different tumors.In addition,the function and mechanism of TRIM33 in lung cancer are not clear at present,especially whether TRIM33 affects the occurrence and development of NSCLC through the function of its E3 ubiquitin ligase.Objective:The function of TRIM33 in non-small cell lung cancer was analyzed by cell phenotype experiments,and it was found that TRIM33 can inhibit the proliferation of NSCLC cells,but its specific mechanism remains unclear.This article will explore the potential mechanism by which TRIM33 regulates cell proliferation through minichromosome maintenance protein 7(MCM7),and further analyze the possible connection between this regulatory mechanism and the acetylation modification of TRIM33.Methods:(1)The expression of TRIM33 in normal lung epithelial cells and NSCLC cell lines was detected by Western Blot.The TCGA and GTEx databases were used to analyze the differential expression of TRIM33 in normal lung tissue and lung adenocarcinoma tissue,and the association of TRIM33 with disease stage and patient survival.(2)Construct TRIM33 overexpression and knockout plasmids,and evaluate cell proliferation and migration of NSCLC cell line NCI-H1299 under TRIM33 overexpression or knockout conditions by cell growth experiments and transwell experiments.The protein levels of N-cadherin,β-catenin,Vimentin,and Snail were detected by Western Blot to examine the EMT.(3)The interaction between TRIM33 and MCM7 was determined by mass spectrometry(MS),co-immunoprecipitation(Co-IP),and immunofluorescence(IF).The effect of overexpression or silencing of TRIM33 on the protein level of MCM7 was detected by Western Blot.Then,the change of ubiquitination level of MCM7 after TRIM33 overexpression was detected by Co-IP.(4)The cells were treated with broad-spectrum deacetylase inhibitors TSA and NAM and detected the acetylation,m RNA,and protein expression levels of TRIM33.(5)TRIM33 protein level was detected after exogenous expression of SIRT1,SIRT6 and SIRT7.The protein interaction between SIRT1 and SIRT6 with TRIM33 was determined by Co-IP,respectively.In addition,the effect of SIRT1 and SIRT6 on the acetylation level of TRIM33 was further detected by Co-IP assay.Finally,Western blot and Co-IP were used to detect whether SIRT1 was involved in the process of TRIM33 regulating MCM7 ubiquitination.Results:(1)TRIM33 expression was significantly downregulated in NSCLC cell lines compared with normal lung epithelial cells.The results of database analysis showed that the expression of TRIM33 was downregulated in lung adenocarcinoma tissues compared with adjacent paracancerous tissues,and the survival rate of NSCLC patients with low expression of TRIM33 was poor.But TRIM33 was not associated with the disease stage.(2)Overexpression of TRIM33 inhibited tumor cell growth,migration,and EMTrelated protein expression.However,the knockdown of TRIM33 promoted cell growth,migration,and EMT-related protein expression.(3)TRIM33 interacts with MCM7 and increases its ubiquitination level,but TRIM33 does not affect the protein stability of MCM7.(4)The deacetylase SIRT inhibitor NAM increased the acetylation level of TRIM33 but had no significant effect on the m RNA and protein expression levels of TRIM33.However,another deacetylase HDAC inhibitor TSA had little effect on both protein expression and acetylation level of TRIM33.(5)The deacetylase SIRT1 and SIRT6 located in cell nucleus interacted with TRIM33,while only SIRT1 can increase the acetylation level of TRIM33.Furthermore,SIRT1 mediated the deacetylation modification of TRIM33 and affected its ubiquitination modification on MCM7.Conclusion:Our study reveals the possible molecular mechanism of TRIM33 inhibiting the proliferation and migration of NSCLC cells.In particular,it is suggested that TRIM33 can regulate its ubiquitination modification of MCM7 through SIRT1-dependent deacetylation,thereby affecting cell proliferation and participating in the occurrence and development of tumors.