| BackgroundGlioma is the most common malignant tumor of the central nervous system.In recent decades,the incidence of primary malignant central nervous system tumors has been increasing,and the incidence of glioma has also increased significantly.At present,the pathogenesis of glioma is not clear.Ionizing radiation,gene variation and signal pathway disorder are the risk factors for glioma.The main treatment for glioma includes surgery and postoperative concurrent chemoradiotherapy.Although,in recent years,target therapy,immunotherapy and biological therapy have made great progress,we still haven't found effective treatment to inhibit the growth of the glioma.Meanwhile,prognosis of glioma is still poor,especially high grade glioma with the median survival 15 months and the 5-year survival rate less than 5%.It is of great significance to investigate the molecular characteristics and biological behaviors of glioma,such as gene expression,gene mutation,single nucleotide polymorphism,gene copy number variation and epigenetic modification for the molecular classification of glioma and the individualized and precise treatment of glioma patients.Non-coding RNAs are a kind of RNA that can be transcribed from the genome but not translated into proteins.Non-coding RNAs have very important biological functions and are involved in the occurrence and development of a variety of diseases.A large number of studies have confirmed that mi RNAs,lnc RNAs and circRNAs with abnormal expression in tumor tissues can be used as molecular markers for tumor diagnosis and prediction of prognosis,as well as providing new therapeutic targets.The abnormal expression of lnc RNA PVT1 is associated with the occurrence and progress of various tumors,including lung cancer,prostate cancer,gastric cancer,colorectal cancer and pancreatic cancer and so on.The previous findings of our team also confirmed that lnc RNA PVT1 also plays an important role in breast cancer,however,the mechanism of lnc RNA PVT1 regultaes tumorigenesis of glioma is still unclear.CircRNAs are involved in regulating a variety of tumor-associated phenotypes and biological processes such as cell cycle and apoptosis,invasion and migration,immunity,angiogenesis and metastasis.However,the specific circRNAs associated with occurrence and development of glioma are unclear.Our previous study has builded expression spectrum of differences circRNAs in glioblastoma through high-throughput sequencing and bioinformatics analysis.We found that circRNA EPB41L5 decreased significantly in glioblastoma and associated with poor prognosis.CircRNA EPB41L5 also regulated the biological behaviors of glioma cell line.However,the internal mechanism remains to be further studied.Part 1.LncRNA PVT1 recruits COPS5 to deubiquitinate and stabilize TRIM24 and promote the occurrence and development of gliomaObjectiveTo investigate the expression feature of lnc RNA PVT1 in glioma and its correlation with prognosis of patients.To study the role of lnc RNA PVT1-COPS5-TRIM24-STST3 regulation axis in tumorigenesis of glioma.Methods1.To study the expression level of lnc RNA PVT1 in glioma and its correlation with prognosis of patients via analyzing GEO and TCGA databases.2.To detect the expression of lnc RNA PVT1 in clinical samples and glioma cell lines by RT-PCR.Knockdown of lnc RNA PVT1 in U251 and U373 to observe its effect on proliferation,clone formation and tumorigenesis of glioma cell.3.The interaction between lnc RNA PVT1 and TRIM24 protein was studied via RNA pull-down assay and RNA Binding Protein Immunoprecipitation assay.WB was used to detect the effect of lncRNA PVT1 on the stability of TRIM24 protein.4.The interaction between TRIM24 and COPS5 was studied via immunoprecipitation assay.Meanwhile,to investigate the role of lnc RNA PVT1 in interaction between TRIM24 and COPS5.5.To study the effect of lnc RNA PVT1 on tumorigenesis of glioma in vivo and related signal pathway.Results1.LncRNA PVT1 is up-regulated significantly in glioma and associated with poor prognosis of glioma patients.2.Knockdown of lnc RNA PVT1 in U251 and U373 inhibit cell proliferation,clone formation and tumorigenesis.3.LncRNA PVT1 interacts with C domain of TRIM24 via exon 5 and exon 6.Overexpression of lnc RNA PVT1 increases expression of TRIM24,meanwhile,lnc RNA PVT1 regulates the stability of TRIM24 protein.4.High throughput screening DUB finds COPS5 interacts with TRIM24 and the expression level of COPS5 is positively correlated with the expression level of TRIM24 protein.The interaction between COPS5 and TRIM24 dependents on LncRNA PVT1.After the deletion of LncRNA PVT1,the interaction between COPS5 and TRIM24 is weakened and TRIM24 ubiquitination level is reduced.5.Down-regulation of lnc RNA PVT1 significantly reduced the expression level of p-STAT3,overexpression of TRIM24 could reverse the down-regulation of p-STAT3 caused by lnc RNA PVT1,and overexpression of TRIM24 could reverse the inhibition of cell proliferation,clone formation and tumorigenesis caused by lnc RNA PVT1.ConclusionLncRNA PVT1 expression level is significantly increased in glioma and associated with poor prognosis of glioma patients.LncRNA PVT1 recruits COPS5 to interact with TRIM24 and form RNA-protein complex which deubiquitinates and stabilizes TRIM24 to increase TRIM24 protein expression at the post-translational level and p-STAT3 expression.LncRNA PVT1-COPS5-TRIM24-STST3 regulation axis plays an important role in the development of glioma.LncRNA PVT1 is of great clinical significance as a biological marker for glioma diagnosis and a potential therapeutic target.Part 2.CircRNA EPB41L5 regulates its host gene EPB41L5 via sponging mi R-19 a to repress glioblastoma tumorigenesisObjectiveCircRNAs are a kind of widely expressed non-coding RNAs in eukaryotic cells and involve in regulating tumorigenesis of many types of cancers.However,the expression profiles and precise function role in glioblastoma remain unclear.Methods1.The expression profiles of circRNAs in glioblastoma were determined by Illumina Hiseq from six glioblastoma tissues and six normal brain tissues.2.Then,we detected the correlation between circRNA EPB41L5 expression and clinical features and survival time of 45 glioblastoma patients.3.RNA-seq was used to find target gene regulated by circRNA EPB41L54.The interaction between circRNA EPB41L5,mi R-19 a and EPB41L5 were tested through luciferase reporter and RNA pull-down assays.5.The effects of ectopic intervention expression of circRNA EPB41L5 or EPB41L5 on proliferation,clone formation,migration and invasion in vitro and tumorigenesis in vivo were observed to evaluate the function of circRNA EPB41L5 or EPB41L5.Results1.CircRNA EPB41L5 was down-regulated in glioblastoma tissues and cell lines compared to normal brain tissues and cell line.Lower circRNA EPB41L5 expression was correlated to poor prognosis of glioblastoma patients.2.Over-expression of circRNA EPB41L5 inhibited proliferation,clone formation,migration and invasion abilities of glioma cells,while suppression of circRNA EPB41L5 had counter effects.3.RNA-seq results determinated that its host gene EPB41L5 was the target gene of circRNA EPB41L5.4.CircRNA EPB41L5 served as a sponge of mi R-19 a and inhibited mi R-19 a activity to up-regulate EPB41L5 expression.5.We found that circRNA EPB41L5 regulated RhoC expression and phosphorylation of AKT through EPB41L5.ConclusionOur study highlights a novel suppressive function of circRNA EPB41L5 and reveals that circRNA EPB41L5-mi R-19a-EPB41L5-AKT regulatory axis plays a striking role in the progression of glioblastoma,which provides a novel insight into the mechanisms underlying development of glioblastoma. |
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