| Part Ⅰ The expression of UBE2T was significantly higher in non-small cell lugn cancer than normal lung tissuesObjective Non-small cell lung cancer is a commen cancer threaten the survial and healthy of humans.The further research about the pathogenesis and the targets to diagnose and treatment is significant to prolong the survivial for patients.Methods The expression profiles of genes were downloaded from the Gene Expression Omnibus and the Cancer Genome Atlas database.The association between UBE2T and overall survival was conducted by the online tool kmplot.The association between UBE2T expression and clinical parameters was explored using the Wilcoxon test by R in the TCGA-LUAD database.Overall survival was explored via the Survival package.Univariate and multivariate Cox analyses were performed to explore the correlation between UBE2T expression,other clinical features and survival.We collected 20 fresh NSCLC samples and adjacent normal lung tissue.The RNAs of these samples were explored by real-time polymerase chain reaction.We further performed an immunohistochemistry study on 10 NSCLC tissue embedded in paraffin.Finally,the western blot was performed in human lung epithelial cell and seven non-small cell lung cancer cells.Results 22 genes showed significant overexpression status in NSCLC tissues.Then,the Kaplan-Meier plotter results showed that UBE2T ranked first in HR and may play an important role in tumorigenesis.The patient with high expression had a worse survival.The expression of UBE2T was associated with tumor size,distant metastases,lymphatic metastasis and tumor stage.The multivariate Cox analysis showed that UBE2T was associated with overall survival and may be a biomarker.UBE2T exhibited higher expression in NSCLC tissues compared to that in their corresponding adjacent normal tissues by qRT-PCR and western blot.The expression of UBE2T was higher in NSCLC cells than that in other cancer cells.Conclusions UBE2T exhibited higher expression in NSCLC tissues compared to that in their corresponding adjacent normal tissues.Part Ⅱ UBE2T promotes cell proliferation and radiation resistance in NSCLCObjective The radiation sensitivity affects the survial for non-small cell lung cancer patients.The exploration for the new targets to enhance the radiation sensitivity is significance.Methods We knocked down and overexpressed UBE2T expression by lentivirus-mediated infection.The cell proliferation and apoptosis were determined by clone formation,EdU incorporation assay and flow cytometric analysis.Then we explored the potential effects of UBE2T on cell migration and invasion using the wound healing and transwell assays.Then,the clone formation assay was used to detect the survival rate under the treatment of radiation.The apoptosis and cell cycle were explored by flow cytometric analysis.Then,the marker of DNA double-strand breaks was also explored by immunofluorescence.We further established a xenograft tumor model in nude mice to explore the further function of UBE2T in vivo.Results The knocked down and overexpressed UBE2T were successfully established and confirmed by western blot and real-time polymerase chain reaction assays.Knockdown UBE2T significantly retarded cell growth compared to the control,whereas overexpression UBE2T accelerated cell growth.UBE2T markedly promoted cell migration and invasion by the wound-healing assay and the transwell assay.Clone formation,flow cytometric and immunofluorescence assays showed that UBE2T knockdown was more sensitive to irradiation and that UBE2T-overexpressing NSCLC cells were more resistant to irradiation.The tumor volumes of mice in the knocked down group were smaller.By X-ray treatment,the difference in tumor volume between the two groups was expanded.Conclusions UBE2T promotes the proliferation,migration,invasion and radiation resistance.The apoptosis rate and the G2/M cell cycle arrest were reduced by UBE2T.Whereas,the silece of UBE2T suppressed the proliferation,migration,invasion and radiation resistance.The apoptosis rate,the G2/M cell cycle arrest and the radiation sensitivity were enhanced by the silence of UBE2T.Part Ⅲ UBE2T promotes the radiation resistance by the ubiquitination of FOXO1 in NSCLCObjective UBE2T exhibited higher expression in NSCLC tissues compared to that in their corresponding adjacent normal tissues.UBE2T promotes the proliferation,migration,invasion and radiation resistance.However,the mechanism remains unclear.It is important to explore the mechanism and downstream pathway to increase the radiotherapy sensitivity of non-small cell lung cancer.Methods To further explore the mechanism of UBE2T in NSCLC,we utilized H1299/vector and H1299/shRNA cells for RNA-Seq analysis.We used the immunoprecipitation method to explore the ubiquitination of FOXO1 by UBE2T.the MG132 was also used to explore the relationship between UBE2T and FOXO1.Then,FOXO1 siRNA was transduced in H1299/shRNA cells to explore the relationship between FOXO1 and radiation sensitivity.The western blot was further used to explore the effect of UBE2T on the downstream pathway of FOXO1.Finally,the effect of UBE2T on the expression of FOXO1 was detected by immunohistochemistry and western blot.Results RNA-Seq showed UBE2T was enrichment in FOXO pathway.UBE2T had no effect on FoxO1 expression on mRNA,but decreased FOXO1 expression on protein level.The expression of FOXO1 was significantly increased in the A549/UBE2T group.The content of ubiquitin by FOXO1 was increased by UBE2T.In combination with radiation,the silence of FOXO1 increased the repair of DNA damage repair,decreased apoptosis and the G2/M cell cycle arrest,and promoted radiotherapy tolerance.Western blot showed that UBE2T activated the Wnt/β-catenin signaling pathway through ubiquitination of FOXO 1,resulting in radiotherapy tolerance.In vivo tumorigenesis experiment showed that UBE2T decreased the expression of FOXO 1 and its downstream pathway protein.Conclusions UBE2T activated the Wnt/β-catenin signaling pathway through ubiquitination of FOXO 1,and promoted radiotherapy tolerance. |
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