Regulation Of The Ubiquitin Ligase TRIM25 On The PTEN/AKT Signaling Pathway And Its Roles In Non-small Cell Lung Cancer

ObjectivesLung cancer is the most common primary malignant tumor with high incidence and high mortality rates.It mainly arises from bronchial mucosal epithelial cells.According to its histological types,lung cancer can be categorized into two classes: small cell lung cancer(SCLC)and non-small cell lung cancers(NSCLC).NSCLC accounts for approximately 80% of the total number of lung cancers,which includes squamous cell carcinoma,adenocarcinoma and large cell carcinoma.Although targeted therapy for lung cancer has made great progress,lung cancer patients remain poor survival rate due to the high incidence of relapse.Therefore,it’s important to explore the function of the oncogene which implies in lung cancer.TRIM25 belongs to the TRIM ubiquitin ligase family,which has been reported in modulating antiviral response and estrogen signal transduction.Recent studies show that TRIM25 is involved in breast cancer,but its roles and mechanism of action in NSCLC are still unclear.The purpose of this study is to explore the mechanism of ubiquitin ligase TRIM25 in NSCLC and to provide more theoretical basis for clinical treatment.Methods1.WB and RT-PCR were applied to detect the protein and message RNA(m RNA)level of TRIM25 in patients’ samples.2.TRIM25 was overexpressed or knocked down in NSCLC cells,and then MTT assay and flow cytometry were applied to detect the function of TRIM25 in NSCLC cell lines,including proliferation and drug resistance.3.TRIM25 was transfected into A549,H460 and H1299 cell lines,and then WB was applied to detect the protein level of cell growth related signal pathway.4.Co-immunoprecipitation(Co-IP)and western blot methods were applied to detect the interaction between TRIM25 and PTEN.5.The truncated TRIM25 plasmids were constructed by molecular cloning method.The interaction between different truncated mutants of TRIM25 and PTEN was identified by Co-IP and WB methods.6.The effects of TRIM25 on the ubiquitin level of PTEN were analyzed by Western bloting and immunoprecipitation.7.The effects of TRIM25 on the stability of PTEN were detected by Western bloting.TRIM25 plasmid was transfected into HEK293 T or NSCLC cells for 36 h,then cells were treated with Cycloheximide(CHX)and cells were harvested in different time.Cell lysates were detected by WB to analyze the half life of PTEN.8.Knocking down PTEN in NSCLC cells and then overexpressing TRIM25,Western bloting was applied to analyze whether the activation of AKT and m TOR induced by TRIM25 was dependent on PTEN.Results1.The protein level and m RNA level of TRIM25 were significantly higher in NSCLC patient’s carcinoma tissue than para-carcinoma tissue.2.TRIM25 was overexpressed or knocked down in different NSCLC cells,MTT assay showed that the proliferation ability of NSCLC cells was positively correlated with the expression of TRIM25.Flow cytometry showed that the survival rate of NSCLC cells was positively correlated with the expression of TRIM25.3.NSCLC cells were transfected with TRIM25 and WB was applied to detect the protein level of PI3K-AKT-m TOR signaling pathway.It was found that TRIM25 activated AKT and m TOR through the upregulated phosphorylation level of AKT and m TOR.Moreover,TRIM25 has no effect on the protein level of PTEN.4.PTEN is an important regulator in PI3K-AKT-m TOR signaling pathway,and TRIM25 has no effect on the protein level of PTEN.In order to determine whether there are any other regulation modes between TRIM25 and PTEN,we found that there was a interaction between TRIM25 and PTEN in HEK293 T,A549 and H460 cell line.5.In order to further determine the binding region between TRIM25 and PTEN,the truncated TRIM25 plasmids were constructed using molecular cloning method.It was found that TRIM25 combined with PTEN through its B-boxes,CC and SPRY domains.6.TRIM25 functions as an E3 ubiquitin ligase can promote the ubiquitination levels of the substrate proteins and regulate their protein levels.Co-transfecting TRIM25 and PTEN into HEK293 T cells,the results showed that TRIM25 could upregulate the ubiquitination level of PTEN without affecting its protein level.7.In order to further determine the effect between TRIM25 and PTEN,CHX experiments showed that TRIM25 has no effect on the half-life of PTEN.8.We knocked down PTEN and then overexpressed TRIM25 into A549,the results showed that the effect of TRIM25 on the proliferation of non-small cell lung cancer cells was dependent on PTEN.ConclusionsIn this study,we demonstrated that the ubiquitin ligase TRIM25 is frequently overexpressed in NSCLC tumor tissues and cancer cell line.TRIM25 promotes the proliferation and drug resistance of NSCLC cells.It was found that overexpression of TRIM25 promoted the phosphorylation on AKT and m TOR in NSCLC cells and induced cell growth and proliferation.We also found that overexpression of TRIM25 can up-regulate the K63-linked ubiquitination on PTEN.When knocked down the endogenous PTEN and then overexpressed TRIM25 in non-small cell lung cancer cells,the phosphorylation on AKT and m TOR was decreased,suggesting that the activation of AKT/m TOR by TRIM25 was dependent on PTEN.The study demonstrated that TRIM25 inhibited the phosphatase function of PTEN by promoting it’s K63-linked ubiquitination,and thereby activating AKT and m TOR,which finally promoted tumorigenesis of non-small cell lung cancer.The study demonstrated that TRIM25 could be used for clinical diagnosis and treatment of patients with NSCLC.