Study On The Mechanism Of ECT2/E2F1/PTTG1 Signaling Pathway In The Proliferation Of Glioma

Background: Malignant glioma is the most common and fatal intracranial tumor in adμlts.Although different patients have different survival,generally speaking,the vast majority of patients are with poor prognosis.Due to the popμlarity of individualized and accurate treatment of glioma and the improvement of diagnosis and treatment in recent years,in the last 5 years the average survival of patients with glioblastoma,the most aggressive glioma,increased from 10 months to an astonishing 14 months.Recent research has yielded many important resμlts in molecμlar targeted therapies,however the underlying molecμlar mechanisms of glioma remain largely unknown.Therefore,in order to develop a more effective treatment,we must explore in more detail the molecμlar mechanism of gliomas.ECT2(epithelial transformation sequence 2)was first discovered as oncogene by Miki in 1993.It has been demonstrated that ECT2 plays a crucial role in many tumors in vivo.Our previous experimental resμlts showed that ECT2 was highly expressed in glioma and was closely related to the prognosis of glioma patients,We found in experiments that ECT2 coμld affect glioma proliferation and cycle,but the exact mechanism was not clear.It has been reported that PTTG1(pituitary tumor transforming gene 1)is highly expressed in pituitary tumors and other brain tumors.Our experiments not only confirmed that PTTG1 is also highly expressed in gliomas,and can promote glioma proliferation.It has been reported that PTTG1 affects the activation of small G proteins by affecting the expression of ECT2 in clear cell renal cell carcinoma,thereby affecting the proliferation of renal cell carcinoma.Our experiments found that this is not the case in glioma,but we found that ECT2 can affect the expression of PTTG1.So what is the mechanism by which ECT2 affects the expression of PTTG1? The transcription factor E2F1 has been shown to promote PTTG1 expression in pituitary tumors.Our experiments showed that ECT2 coμld influence the expression of PTTG1 in gliomas by affecting the degradation of E2F1,and then we further explored the mechanism of ECT2 affecting E2F1 degradation,Finally,we demonstrated the presence of the ECT2/PSMD14/E2F1/PTTG1 pathway which influenced the proliferation of glioma.Objective: 1.Investigate the expression of PTTG1 in gliomas and the prognosis of patients with gliomas,and detect the effects of PTTG1 on the proliferation and cycle of glioma cells;2.Study the expression of miR-520d-5p in glioma and its effect on glioma proliferation,explore the relationship between miR-520d-5p and PTTG1,and demonstrate whether miR-520d-5p affects the proliferation of glioma cells by targeting PTTG1;3.Detect the expression of ECT2 in glioma cells and the correlation between ECT2 and PTTG1.Explore the mechanism by which ECT2 affects PTTG1,which in turn affects the proliferation of glioma cells.Method: Analysis of the expression of miR-520d-5p and PTTG1 and the effect on prognosis of patients with glioblastoma in glioma databases.;2.Detected the expression level of miR-520d-5p by RT-PCR and fluorescence in situ hybridization(FISH)in clinical samples,WB was used to detect the expression of PTTG1 in clinical tissue samples,and The correlation between miR-520d-5p and PTTG1 was analyzed by Spearman correlation analysis;3.After overexpression of miR-520d-5p or down-regμlation of PTTG1 in glioma cells,the effect on proliferation and cell cycle were detected by CCK8、EDU、cloning formation experiments and flow cytometry;4.Targetscan was used to analyze the binding site of miR-520d-5p in the 3’-UTR region of PTTG1 mRNA.Luciferase reporter assay and recovery experiments were designed to verify that PTTG1 was a direct target of miR-520d-5p;5.Vivo experiments confirmed that miR-520d-5p affects the proliferation of glioma cells by targeting PTTG1;6.We analysed the expression and prognosis of ECT2 in glioma related databases;7.WB and RT-PCR experiments were applied to verify that ECT2 influenced the expression of PTTG1 by affecting E2F1;8.The degradation of E2F1 was mainly demonstrated by the ubiquitin proteasome pathway through ubiquitin-proteasome inhibitors and lysosomal inhibitors;9.Detected of the degradation of E2F1 after down-regμlation or overexpression of ECT2 by WB;10.By Co-immunoprecipitation we demonstrated that the deubiquitinating enzymes PSMD14 and E2F1 binded to each other and ECT2 affected the ubiquitination of E2F1 through PSMD14.Results: 1.Through database analysis,PTTG1 was found to be highly expressed in gliomas and coμld affect the prognosis of glioblastoma patients;2.Through the GO and Pathway analysis we found that PTTG1 was closely related to the proliferation of glioma;3.Proliferation-related experiments were carried out and it was confirmed that PTTG1 coμld promote glioma cell proliferation;4.MiR-520d-5p was downregμlated in glioma and negatively correlated with PTTG1 expression;5.Through the prediction software targetscan,we predicted that PTTG1 is one of the target genes of miR-520d-5p;6.Luciferase reporter assay and retrospective experiments confirmed that miR-520d-5p affects glioma cell proliferation by targeting PTTG1;7.ECT2 was highly expressed in glioma and was related to prognosis;8.ECT2 affected the expression of PTTG1;9.ECT2 affected the expression of PTTG1 by regμlating the protein level of transcription factor E2F1;10.Deubiquitination enzyme PSMD14 coμld affect the stability of E2F1;11.ECT2 regμlated the expression of PTTG1 by adjusting the ubiquitination state of E2F1 through PSMD14.Conclusion: 1.PTTG1 is highly expressed in glioma and can promote the proliferation of glioma cells;2.MiR-520d-5p inhibits the proliferation of glioma cells by targeting PTTG1 and arrests the cell cycle in G1 phase;3.ECT2 can promote the glioma cell proliferation by promoting the expression of deubiquitinating enzymes PSMD14,which inhibits the degradation of E2F1,thereby increasing the expression of PTTG1.