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The Role And Mechanism Of UBQLN2 In The Proliferation And Invasion Of Glioma

Posted on:2021-07-15Degree:MasterType:Thesis
Country:ChinaCandidate:M J LiuFull Text:PDF
GTID:2544307160985239Subject:Oncology
Abstract/Summary:PDF Full Text Request
Background and Objective:Glioma is the most common primary tumor of the adult central nervous system,accounting for about 80% of malignant brain tumors.The origin and pathogenesis of its disease are still unclear.Glioblastoma(GBM)is the most malignant type of glioma,the incidence of which accounts for 50% of the entire glioma,and the average survival time of patients after receiving standard treatment is only 12-15 Months.Glioma is belong to aggressive growth pattern that is easy to invade the surrounding brain tissue,which limits the eradication of surgery and is a key factor leading to recurrence and affecting prognosis.To studying and exploring the invasive growth characteristics of gliomas,and grasping the molecular mechanisms of their occurrence and development,are it is great significance for in-depth understanding of the lethal mechanism of such tumors,guiding drug development and exploring effective tumor treatment methods.In the early stage of our laboratory,we screened glioblastoma cells in vitro through the CRISPR-Cas9 whole gene knockout library,and found that multiple genes are closely related to the growth of glioblastoma.UBQLN2 is one of the more significant genes.UBQLN2 is a member of the ubiquilin family of proteasome shuttle factors,which is mainly located in the cytoplasm and has a small distribution in the nucleus.It plays an important role in various biological processes such as protein ubiquitination degradation,lysosomal homeostasis and autophagy,cell signaling,and endoplasmic reticulum stress.The UBQLN2 gene is located on the X chromosome p11.21,and its protein has ubiquitin-like domain(UBL)and ubiquitin-associated domain UBA at the N-terminus and C-terminus,respectively,which are connected to proteases.And ubiquitinated substrates to target ubiquitinated proteins to the proteasome for degradation.In addition,the middle region of the N-terminus and C-terminus of the UBQLN2 protein also includes multiple heat shock chaperone protein binding(STI)motifs and multiple PXX repeat sequence domains,and their functions have not been elucidated.UBQLN2 is also involved in the formation of autophagosomes and promotes the maturation of autophagy-related protein LC3 from the cytoplasmic form LC3Ⅰ to the membrane-bound form LC3Ⅱ.Current research focuses on the role of UBQLN2 mutations in amyotrophic lateral sclerosis(ALS)and neurodegenerative diseases.UBQLN2 has been reported to inhibit non-small cell lung cancer cell metastasis and epithelial mesenchymal transformation,but the biological function and role of UBQLN2 in the occurrence and development of gliomas are not yet clear.In this study,the clinical tissue specimens and cell lines of glioma were taken as the research objects.It was initially found that the expression of UBQLN2 was significantly down-regulated in cancer tissues and glioma cell lines.It was further planned to construct T98 G,U251 overexpressing UBQLN2 and U251 knock down UBQLN2 cell model,to explore the role and mechanism of UBQLN2 in the development of glioma from the in vivo and in vitro levels,and to analyze the correlation between the expression of UBQLN2 and the clinical parameters of glioma,in order to explain UBQLN2 as a glioma treatment Potential targets and prognostic markers provide important theoretical basis.Research methods:1.Analyze the expression of UBQLN2 in gliomas using online clinical data platforms TCGA,REMBRANDT,and CGGA.q RT-PCR was used to detect the expression of UBQLN2 in 10 glioma tissues and 27 normal tissue samples.Western bolt was used to detect the protein expression of UBQLN2 in 12 pairs of glioma tissues and matched normal tissue samples.The Cbioportal database analyzes the UBQLN2 gene structure of patients with glioblastoma.2.q RT-PCR and Western blot detect the expression of UBQLN2 in different glioma cells.The lentivirus constructing UBQLN2 recombinant plasmid was constructed by infecting glioma cells T98 G and U251 to construct the overexpressing UBQLN2 stable strain,and the overexpression efficiency of the overexpressing UBQLN2 cell line was detected by GFP fluorescence intensity,q RT-PCR and Western blot.The U251 cell UBQLN2 gene model was knocked out using CRISPR/Cas9.The sequencing verification,q RT-PCR and Western blot were used to detect the knockdown efficiency of U251 cells.The effects of UBQLN2 on the growth and proliferation of glioma cells were studied using EDU,plate cloning,and cell cycle in vitro experiments.U251/UBQLN2 and U251/NC cells with Luciferase labeling,U251KO22#,U251KO32# and U251 cells knocked down UBQLN2 were inoculated into nude mice in situ to establish nude mice in situ tumor model,treated with Luciferase luminescent substrate,applied Animal in vivo imaging was used to observe the effect of overexpression of UBQLN2 on tumor formation in nude mice.Pathological analysis was performed on the in situ tumor model of nude mice by HE staining and immunohistochemistry ki-67 staining to observe the proliferation of tumor cells.In vivo study of UBQLN2 on glioma Effects of proliferation.In UBQLN2 overexpressing cell lines T98G/UBQLN2,U251/UBQLN2 and control cell lines T98G/NC,U251/NC,the changes of autophagy-related molecules LC3Ⅰ and LC3Ⅱwere detected by Western blots,and the effect of UBQLN2 on cell autophagy was analyzed.Construct the dual-fluorescence autophagy flow GFP-LC3-RFP-m LC3 lentiviral plasmid,package the lentivirus and infect U251,U251KO32# cells,and analyze the effect of knocking down UBQLN2 on glioma autophagy flow by using live cell workstation.At the same time,flow cytometry was used to detect the changes of autophagic flow in U251 and U251KO32# cells.Western blot detected the phosphorylation level of m TORC1 target P70 and ULK1 in m TOR signaling pathway.Immunohistochemistry was used to detect the phosphorylation levels of m TORC1 target P70 and ULK1 in the m TOR signaling pathway.3.The effects of UBQLN2 on invasion and metastasis of glioma cells were detected by cell scratch test and Transwell in vitro test.HE staining and immunohistochemistry were used for pathological analysis and detection of EMT related markers in nude mice orthotopic tumor models,and to explore the effect of UBQLN2 on glioma invasion and metastasis in vivo.Western blot detect the expression of EMT related molecules.4.Clinical data platform of glioma samples to analyze the relationship between UBQLN2 and the prognosis of glioma patients.Record the survival time of the nude mouse orthotopic tumor model after inoculation of tumor cells,and draw a survival curve.Immunohistochemistry was used to detect the expression of UBQLN2,ZEB1,N-cadherin,vimentin,ki-67,P-P70,P-ULK1 in human glioma tissues and tissues adjacent to cancer.The UBQLN2 expression and the clinical status of glioma patients were analyzed through case data Relevance of features.Result:1.Analysis of glioma sample database The expression of UBQLN2 in glioma tissue is lower than that in normal brain tissue.In gliomas of different clinicopathological types,the expression of UBQLN2 in glioblastoma with the highest degree of malignancy was significantly lower than that of other types.Analysis of the chromosomal structure of glioma tissues indicates that some patients with glioblastoma have UBQLN2 gene deletion.2.Successfully constructed a stable glioma cell model with overexpression and knockdown of UBQLN2.It was found that overexpression of UBQLN2 can inhibit the proliferation of glioma cells in vitro,and knockdown of UBQLN2 can promote the proliferation of glioma cells in vitro.The glioma cells overexpressing UBQLN2 have reduced tumorigenic ability in vivo,and the glioma cells knocking down UBQLN2 have increased tumorigenic ability in vivo.Immunohistochemistry ki-67 staining showed that overexpression of UBQLN2 slowed down the proliferation ability of the control group,knocking down UBQLN2 enhanced the proliferation ability of the control group.Western bolt detection overexpression of UBQLN2 in glioma cells significantly activated autophagy and promoted cell catabolism compared with the control group.At the same time,the dual fluorescence autophagy flow detection system also showed that knocking down UBQLN2 can inhibit the autophagy flow of U251 cells.Western blot results showed that after overexpression of UBQLN2,the phosphorylation levels of P-70 and ULK1 were significantly reduced.Immunohistochemical results showed that the expression levels of P-P70 and P-ULK1 decreased after overexpression of UBQLN2,and the expression levels of P-P70 and P-ULK1 increased after knocking down UBQLN2.3.The in vitro cell test showed that overexpression of UBQLN2 can inhibit the migration and invasion of glioma cells,and knocking down UBQLN2 can promote the migration and invasion of glioma cells.Meanwhile,Western bolt results showed that overexpression of UBQLN2 in glioma can inhibit the expression of EMT signaling factor.The results of HE staining and immunohistochemistry showed that the invasion ability of U251 cells overexpressing UBQLN2 in vivo decreased,the expression of Vimentin,N-cadherin,and ZEB1 decreased,and the invasion ability of U251 cells increased after knocking down UBQLN2,and the expression of Vimentin,N-cadherin,and ZEB1 increased.increase.4.Survival analysis of the glioma sample database found that the higher the expression of UBQLN2 in glioma patients,the better the survival prognosis(p<0.0001).The in situ model experiment of nude mice also showed that the survival rate of mice decreased after knocking down UBQLN2(p=0.0021,p=0.0024).Analysis of clinical glioma tissue specimens found that the expression of UBQLN2,Vimentin,ZEB1,N-cadherin,ki-67,P-P70,P-ULK1 in gliomas was higher than that in distant paracancerous tissues.Through case data analysis,the expression of UBQLN2 was related to the gender,ZEB1 and N-cadherin expression of glioma patients.Conclusion:1.The expression of UBQLN2 is low in gliomas,especially in glioblastomas with the highest degree of malignancy,which is significantly lower than other types;chromosomal deletion may be part of the reason for the decreased expression of UBQLN2 in gliomas.2.UBQLN2 activate cell catabolism and inhibit anabolic metabolism by promoting autophagy to inhibit the proliferation of glioma cells.3.UBQLN2 inhibit the invasion and metastasis of glioma cells by inhibiting EMT.4.UBQLN2 can be used as a prognostic marker for glioma.
Keywords/Search Tags:glioma, UBQLN2, autophagy, EMT, proliferation, invasion and metastasis
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