| BackgroundThe treatment of patients with glioblastoma(GBM)remains to be one of the greatest challenges in all of oncology.As the most common and lethal primary brain malignancy in adults,it often exhibits radial expansion and infiltrative growth nature,standard of care,including maximum surgery with postoperative concurrent radiotherapy and chemotherapy,leads to median survival time of only 12-15 months and the 5-year survival rate of less than 10%.With deeper understanding of molecular pathology and cancer genome in GBM,many intracellular signaling pathways involved in malignant biological processes of tumors have been found dysregulated,such as PI3K/AKT,RB,p53 signaling pathways.The clinical trial results suggested that the current molecular approaches targeting these key aberrant signaling pathways has limited benefits,which indicated that GBM is of highly heterogeneity,and its progression and recurrence cannot be controlled by a single signaling pathway or molecule.Therefore,finding out key molecules regulating GBM signaling will help to advance our knowledge of GBM progression and recurrence,explore new molecular targeted therapies,and solve the current predicament of precision therapy for GBM.Nuclear factor kappa-light-chain-enhancer of activated B cells(NF-κB)is a family of homo or heterodimeric DNA binding complexes that can control DNA transcription and cytokine production,which is composed of five structurally related members by combination,including p50(also named NF-κB1 or p105),p52(also named NF-κB2 or p100),p65(also named RelA),RelB and c-Rel,which usually exists in the cytoplasm and is inactivated by interacting with IκB proteins(Inhibitor of NF-κB)to form a protein trimer.When the upstream signal factor stimulates the targeted receptor and will activate the IKK(IκB kinase)complex,leading to phosphorylation and subsequent degradation of IκB proteins,which help NF-κB dissociate from the trimer and enter the nucleus to promote downstream gene transcription by exposing nuclear localization motif(NLM).Recent studies have reported that aberrant activation of NF-κB may not only be related to inflammation and immune system diseases,but also play a crucial role in progression of various human cancers,including GBM,by promoting tumor cells proliferation,blood vessel formation,and treatment resistance.Although the contribution of NF-κB signaling pathway to the malignant phenotypes of GBM is well-established,such as rapid growth and radio/chemo-resistance,molecular approaches targeting this pathway are still non-specific or specific targeting IKK that induce a broad range of cellular responses,which has limited survival benefits for GBM patients compared with standard therapy.Therefore,exploring the underlying mechanism of abnormal activation of NF-κB in GBM cells,and analyzing the gene regulatory network of this pathway may help to develop specific small molecule inhibitors in different ways and move current therapeutic strategies forward.Long non-coding RNA(lncRNA)is a type of non-coding RNAs with a length of more than 200 nucleotides(nt)with various cellular functions.With the rapid development of next-generation sequencing technology,a huge number of novel lncRNAs have been identified.Because the number of non-coding genes far exceeds that of protein-encoding genes,it is expected to provide a broad view for discovering disease-related predictive biomarkers for diagnosis/prognosis and therapeutic targets.LncRNAs can not only participate in chromosome silencing,genome imprinting,chromatin modification,transcription activation and interference to regulate gene expression,possibly by interacting with protein,RNA or DNA to achieve biological functions.It has been reported that lncRNAs can affect the angiogenesis,invasion and migration,rapid growth,and treatment resistance of various human cancers,including GBM,by modulating disease-related core signaling pathways,thereby promoting or inhibiting tumor progression.Increasing evidence have shown that a subgroup of lncRNAs is closely related to the NF-κB signaling pathway apart from proteins and miRNAs.For instance,the expression of NKILA(NF-κB interacting lncRNA)in breast cancer cells can be regulated by NF-κB transcriptional activity which can also inactivate NF-κB signaling in turn through a negative feedback loop by inhibiting IκBα phosphorylation;MALAT1(Metastasis-associated lung adenocarcinoma transcript 1)can interact with the p65-p50 protein complex and decreasing its binding to specific DNA elements,thereby inhibiting the transcription activity of NF-κB.The crosstalk between IncRNA-NF-κB has attracted many researchers’attention in recent years.SChLAP1(Second chromosome locus associated with prostate-1;LINC00913),as a member of lncRNAs,has been reported to play an oncogenic role in prostate and bladder cancer by regulating proliferation and metastasis of tumor cells and serve as a prognostic biomarker.Preliminarily we used qRT-PCR to detect the expression level of SChLAP1 in normal brain and glioma tissues(WHO Ⅱ-Ⅳ),and found that it preferentially expressed in high grade gliomas,suggesting SChLAP1 may be related to glioma development and progression.However,its correlation with molecular markers of gliomas,such as IDH and ATRX mutation remains to be elucidated.In addition,the tumor-promoting role of SChLAPl in GBM cells and underlying mechanism are still unknown and require further investigations.Ubiquitination is a commonly studied form of post-translational modification(PTM),which can guide protein function changes and decide its fate,and thereby affect several aspects of cellular physiology.Lys-48(K-48)and Lys-63(K-63)in ubiquitin are the most common and abundant polyubiquitin chain attachment sites to substrates:K-48 ubiquitin chain is a sign that the substrate is recruited to the 26S proteasome for degradation,while the K-63 ubiquitin chain plays a vital role in enzyme activity change,signal transduction and endocytosis.The K-48 and K-63 polyubiquitination modification can mediate the activity of the canonical and non-canonical NF-κB signaling pathways by affecting the expression and function of key components.The ubiquitination reaction process requires a series of enzymes,such as ubiquitin activating enzyme E1,ubiquitin conjugating enzyme E2,ubiquitin ligase E3.Abnormality in enzymatic cascade may lead to signaling change,and even occurrence of diseases such as tumors.Although broad targeting E1 and E2 are possible,E3s are particularly attractive targets because they confer high specificity,and the number is much higher.TRIM(Tripartite motif)family of proteins,structurally characterized by having a cluster of domains starting with an N-terminal RING domain,followed by one or two B box domains,and then a coiled-coil domain,has been extensively linked to NF-κB transcriptional activity through their E3 ubiquitin ligase activity in a variety of human normal cells.However,which TRIM family of gene can regulate the transcriptional activity of NF-κB in GBM cells and regulatory mechanism of action is as yet unclear,and further investigation is required.A variety of biological techniques have been utilized in this study to investigate the tumor-promoting effects of lncRNA SChLAP1 and E3 ligase TRIM22 and regulation of NF-κB transcription activity in GBM cells,which will shed light on novel therapeutic targets that can be developed.Part Ⅰ:LncRNA SChLAP1 forms a growth-promoting complex with HNRNPL in human glioblastomaObjectiveTo reveal the expression level of lncRNA SChLAP1 in normal brain and glioma tissues and cellular distribution of SChLAP1 in GBM cell lines.To analyze the correlation between SChLAP1 expression level and clinicopathological factors of glioma.To explore the growth-promoting effects of SChLAP1 on GBM cells in vitro and in vivo.To identify specific interacting proteins of SChLAP1,and clarify the biological function of the interaction and the effects on downstream signaling.Methods1.RNA in situ hybridization(RNA-ISH)was performed to detect the expression level of SChLAP1 in normal brain and glioma tissues;RNA fluorescence in situ hybridization(RNA-FISH)and qRT-PCR on nuclear and cytoplasmic cellular subfractions were performed to evaluate cellular distribution of SChLAP1 in Normal Human Astrocytes(NHAs)and GBM cell lines LN229,U118MG,GBM#P3 and BG7.2.qRT-PCR was performed to confirm the efficiency of SChLAP1 knockdown or exogenous overexpression lentiviruses;cell number counting and colony forming assays were used to evaluate the proliferative capability of GBM cells in vitro;orthotopic xenograft models were established,and in vivo bioluminescence imaging system was used to monitor the growth of glioma cells in vivo;Kaplan-Meier survival curve was used to analyze survival time of tumor-bearing mice.3.RNA pull-down followed by LC-MS/MS were utilized to identify interacting protein candidates of SChLAPl in GBM cells;RNA immunoprecipitation assay(RIP)was performed to verify the interaction between SChLAP1 and HNRNPL;RNA pull-down assay was used to explore the functional domains responsible for SChLAP1-HNRNPL complex formation.4.Co-immunoprecipitation(Co-IP)followed by LC-MS/MS were utilized to screen out ACTN4 as a downstream effector of the SChLAP1-HNRNPL complex;immunohistochemical staining(IHC)was performed to analyze ACTN4 protein level in normal brain and glioma tissues;Kaplan-Meier survival curve was used to analyze survival time of glioma patients based on ACTN4 expression level in Rembrandt and CGGA databases;Co-IP was performed to identify the functional domains responsible for ACTN4-HNRNPL interaction.5.The indicated GBM cells were pretreated with proteasome inhibitor MG132(20μM)and protein synthesis inhibitor Cycloheximide(CHX;25μg/mL)followed by Western blot to detect half-life of ACTN4 protein in response to SChLAP1;in vivo ubiquitination assay was performed to detect total polyubiquitination levels of ACTN4 protein;Co-IP was performed to evaluate the interaction between ACTN4-HNRNPL in response to SChLAP1 after incubation with MG 132 for 8h.6.Cluster heatmap analysis using CGGA database was performed to show the relationship between ACTN4-associated genes and representative clinical features;Venn diagram was used to illustrate common genes between ACTN4-correlated genes and Rel/NF-κB target genes;luciferase reporter assay was performed to detect the transcriptional activity of NF-κB;Immunofluorescence(IF)and Western blot on nuclear and cytoplasmic cellular subfractions were used to analyze the subcellular localization of P65 protein.Results1.LncRNA SChLAP1 is most abundantly expressed in GBM tissues and mainly localized to the nucleus of GBM cells.Results from RNA-ISH assay showed that expression of SChLAP1 in normal brain tissues was nearly absent.Compared with low-grade gliomas(LGG,WHO grade Ⅱ),SChLAP1 was preferentially expressed in high-grade gliomas(HGG,WHO Ⅲ-Ⅳ),and most abundantly expressed in GBM.Increased expression of SChLAP1 significantly correlated with advanced WHO grade and wild-type IDH1.Results from RNA-FISH showed that fluorescence intensity of Cy3-SChLAP1 detected in GBM cell lines LN229,U118MG,GBM#P3 and BG7 were higher than that in NHAs.qRT-PCR on nuclear and cytoplasmic fractions of RNA obtained from these cell populations was performed to show that SChLAP1 was mainly expressed in the nucleus,while lesser in the cytoplasm.2.Knockdown of SChLAP1 inhibits growth of GBM cells in vitro and in vivoResults from qRT-PCR showed that SChLAP1 expression was reduced by two short hairpin RNAs(shRNAs)targeting SChLAP1 in LN229 and GBM#P3 cells.Cell number counting and colony forming assays were used to demonstrate that SChLAP1 knockdown attenuated proliferation of GBM cells significantly.Through monitoring tumor growth using in vivo bioluminescence imaging system in orthotopic xenograft models,SChLAP1 knockdown in GBM cells was shown to defer the growth significantly in vivo and prolong median survival time of tumor-bearing mice compared with the control tumors.3.HNRNPL is identified as a protein partner of SChLAP1 in GBM cellsResults from RNA pull-down followed by LC-MS/MS analysis identified that HNRNPL is one of interacting protein candidates of SChLAP1.RIP assay further confirmed the physical association between SChLAP1 and HNRNPL.RRM2 of HNRNPL and exon 2(338-433 bp)of SChLAP1 were key to the formation of the SChLAP1-HNRNPL complex.In addition,manipulation of SChLAP1 levels had no effect on HNRNPL protein levels,while HNRNPL knockdown led to reduced stability of SChLAP1,suggesting that HNRNPL stabilized SChLAP1 through the generation of a protein-RNA complex and might be a critical functional partner of SChLAP1 in GBM cells.4.SChLAP1 promotes association between HNRNPL and ACTN4Co-IP followed by LC-MS/MS analysis screened out ACTN4 as one of the downstream effector candidates of the SChLAP1-HNRNPL complex.Results from two-way co-IPs demonstrated that ACTN4 was a protein partner of HNRNPL in GBM cells.The Rod and CaM-like domains of ACTN4 and the RRM3 domain of HNRNPL were critical elements for complex formation between ACTN4 and HNRNPL.Results from IHC on tissue microarray revealed increased ACTN4 to be associated with increased age,wild type IDH1,positive ATRX staining and increased tumor grade.Kaplan-Meier survival curve using Rembrandt and CGGA databases suggest that greater expression of ACTN4 was also correlated with poorer overall survival in patients.5.The SChLAP1-HNRNPL complex inhibits ubiquitination and proteasomal degradation of ACTN4Expression correlation analysis conducted in GBM cell lines and tissue microarrays demonstrated that levels in SChLAP1 and ACTN4 protein were coordinately regulated.Changes in ACTN4 mRNA levels were insignificant regardless of the expression levels of SChLAPl in GBM cells.However,on western blot,levels of ACTN4 changed significantly in response to loss or gain of SChLAP1,but ACTN4 levels can be restored after MG132 treatment,indicating that SChLAP1 may inhibit the proteasomal-ubiquitination degradation of ACTN4.Cycloheximide chase and in vivo ubiquitination assay were performed to confirm above results.Results from Co-IP showed that knockdown of SChLAP1 led to reduced binding of HNRNPL to ACTN4 in LN229 cells,while overexpression promoted association between the 2 proteins.6.SChLAP1 activates NF-κB signaling,a downstream signaling of ACTN4Cluster heatmap analysis using CGGA database showed 1046 positive and 668 negatively correlated genes of ACTN4,and increased expression of ACTN4 also correlated with increasing tumor grade,classical TCGA subtype,older age,and wild type IDH1 status.In addition,by comparing the public database of NF-κB target genes(http://bioinfo.lifl.fr/NF-KB/)with ACTN4 correlated genes,we found 19 genes to be overlapping between these 2 groups;Results from luciferase reporter assay showed that knockdown of SChLAP1 attenuated the transcriptional activity of NF-κB in GBM cells,while increased by overexpression of SChLAP1.Immunofluorescence(IF)and Western blot on nuclear and cytoplasmic cellular subfractions revealed the positive correlation between expression of SChLAP1 and nuclear translocation of P65 protein.7.SChLAP1 promotes GBM growth dependent on interaction with HNRNPLCell number counting assay showed that the enhanced proliferation of GBM cells in vitro by SChLAP1 overexpression was abolished by SChLAP1-exon2 deletion or HNRNPL silencing.Through monitoring tumor growth using in vivo bioluminescence imaging system in orthotopic xenograft models,SChLAP1 overexpression in GBM cells was shown to accelerate the growth significantly in vivo and shorten median survival time of tumor-bearing mice compared to the control tumors,which could also be abrogated by SChLAP1-exon2 deletion or HNRNPL silencing.Conclusion1.SChLAP1 is preferentially expressed in HGG,and positively correlated with several clinicopathological factors;2.SChLAP1 forms a tumor-promoting complex with HNRNPL,which acts a key downstream effector;3.SChLAP1 enhances the binding of HNRNPL with ACTN4,and reduces the proteasomal-ubiquitination degradation of ACTN4 protein to increase protein stability;4.SChLAP1 drives the nuclear translocation of P65 protein to activate the NF-κB signaling pathway via ACTN4 protein,thereby leading to malignant proliferation of GBM cells;Part Ⅱ:TRIM22 promotes glioblastoma cell proliferation via its E3 ligase activity by activating NF-κB signalingObjectiveTo screen and identify most likely genes of TRIM family involved in activating NF-κB signaling in GBM cells.To reveal its expression level in normal brain and glioma tissues.To analyze the correlation between its expression level and clinicopathological factors of glioma.To explore the growth-promoting effects of this gene on GBM cells in vitro and in vivo,and the molecular mechanism underlying activation of NF-κB signaling pathway.Methods1.NF-κB luciferase reporter assay was introduced to screen genes of TRIM family involved in activating NF-κB signaling in U87MG and LN229 cells.2.IHC was performed to detect expression level of TRIM22 in normal brain and glioma tissues.3.Co-IP was performed to verify the interactions between TRIM22-IκBα and TRIM22-IKKγ.4.Western blot was performed to verify the knockout or exogenous overexpression efficiency of TRIM22 lentiviruses;cell number counting and IF for Ki-67 were used to evaluate the proliferative capability of GBM cells in vitro;luciferase reporter assay was performed to detect the transcriptional activity of NF-κB;IF and Western blot on nuclear and cytoplasmic cellular subfractions were used to analyze the subcellular localization of P65 protein;orthotopic xenograft models were established,and in vivo bioluminescence imaging system was used to monitor the growth of glioma cells in vivo;Kaplan-Meier survival curve was used to analyze survival time of tumor-bearing mice.5.The indicated GBM cells were pretreated with proteasome inhibitor MG132(20μM)and protein synthesis inhibitor Cycloheximide(CHX;25μg/mL)followed by Western blot to detect half-life of IκBα protein in response to TRIM22 knockout or wild type/mutant overexpression;in vivo and in vitro ubiquitination assays were performed to evaluate K48-linked polyubiquitination levels of IκBα and K63-linked polyubiquitination levels of IKKy in response to TRIM22 knockout or wild type/mutant overexpression.Results1.Identification of TRIM22 as a positive regulator of NF-κB signaling and a predictor for higher grade of human glioma malignancy.Results from NF-κB luciferase reporter assays showed that among the five genes of TRIM family(TRIM5,TRIM21,TRIM22,TRIM38,TRIM56),only knockdown of TRIM22 decreased transcriptional activity of NF-κB significantly in U87MG and LN229 cells;Expression of TRIM22 in normal brain tissues was nearly absent.TRIM22 was preferentially expressed in HGG and most abundantly expressed in GBM.Increased expression of TRIM22 significantly correlated with wild-type IDH1,wild-type ATRX and advanced WHO grade.2.Knockout of TRIM22 inhibits GBM cell growthResults from Western blot showed that TRIM22 expression was reduced by two small guide RNAs(sgRNAs)in U87MG and LN229 cells expressing Cas9;The key components of NF-κB signaling pathway,phosphorylation levels of IKKα/β(Ser176/180),IκBα(Ser32/36),and P65(Ser536)were uniformly decreased without affecting the total protein levels,except in the case of IκBα protein.Results from cell number counting and IF for Ki-67 demonstrated that TRIM22 knockout attenuated proliferation of GBM cells significantly.Through monitoring tumor growth using in vivo bioluminescence imaging system in orthotopic xenograft models,TRIM22 knockout in GBM cells was shown to slow down the growth significantly in vivo and prolong median survival time of tumor-bearing mice compared to the control tumors.3.The intrinsic E3 ligase activity is critical for TRIM22-mediated cell growth in GBM cellsResults from Western blot showed that phosphorylation levels of IKKα/β(Ser176/180),IκBα(Ser32/36),and P65(Ser536)were uniformly elevated,while total IκBα protein was decreased in LN229 and U118MG cells overexpressing TRIM22-full length(Flag-TRIM22-FL).However,each of these proteins remained unchanged in cells overexpressing two E3 ligase defective mutants(Flag-TRIM22-ΔRING/-TRIM22-C15/18A).Cell number counting and IF for Ki-67 showed that overexpression of TRIM22(Flag-TRIM22-FL)significantly increased proliferation of GBM cells in vitro.Through monitoring tumor growth using in vivo bioluminescence imaging system in orthotopic xenograft models,overexpression of TRIM22(Flag-TRIM22-FL)in GBM cells was shown to accelerate the growth significantly in vivo and shorten median survival time of tumor-bearing mice compared to Flag-EV groups,but remained stable in E3 ligase defective groups(Flag-TRIM22-ΔRING/-TRIM22-C15/18A).In addition,results from luciferase reporter assay revealed that NF-κB signaling activity increased significantly in GBM cells overexpressing TRIM22(Flag-TRIM22-FL)compared with Flag-EV groups,but remained unchanged in cells expressing Flag-TRIM22-ΔRING/-TRIM22-C15/18A.Elevated nuclear accumulation of P65 in GBM cells overexpressing TRIM22(Flag-TRIM22-FL)was detected by IF and Western blot on nuclear and cytoplasmic cellular subfractions compared with Flag-EV groups,but still remained unchanged in Flag-TRIM22-ΔRING/-TRIM22-C15/18A groups.4.TRIM22 regulates IκBα stability via ubiquitin-proteasomal degradation pathwayFurther studies showed that modulating TRIM22 levels increased the IκBα protein levels without affecting IκBα mRNA,but remained unchanged in cells overexpressing E3 ligase defective mutants(Flag-TRIM22-ΔRING/-TRIM22-C15/18A),indicating that TRIM22 might regulate IκBα protein by proteasomal-ubiquitination degradation pathway.Results from cycloheximide chase assay revealed that the half-life of IκBα protein was prolonged by TRIM22 knockout,while shortened by TRIM22 overexpression(Flag-TRIM22-FL),but changes were not significant by E3 ligase defective mutants.Results from in vitro/vivo ubiquitination assays further demonstrated it.Co-IP was performed to show the interaction between TRIM22 and IκBα proteins,which relies on 89-131aa(B-Box domain),133-223aa(Coiled-coil domain),352-498aa(SPRY domain)of TRIM22 and 182-317aa(AR4/AR5)of IκBα for complex formation.5.TRIM22 modulates K63-Iinked ubiquitination levels of IKKy,the regulatory subunit of IKK complex,through protein-protein interactionCo-IP was performed to verify the interaction between TRIM22 and IKKy in GBM cell lines;Results from in vivo ubiquitination assay showed that K63-linked polyubiquitination levels of IKKγ were decreased by TRIM22 knockout,while increased by TRIM22 overexpression(Flag-TRIM22-FL),but changes were not significant by E3 ligase defective mutants(Flag-TRIM22-ΔRING/-TRIM22-C15/18A).6.IκBα ediates the tumor growth-promoting effects of TRIM22Luciferase reporter assay showed that enhanced transcriptional activity of NF-κB by overexpression of TRIM22(Flag-TRIM22-FL)was abrogated by srIκBα overexpression in GBM cells.Cell number counting assay showed that increased proliferation of GBM cells by overexpression of TRIM22 was inhibited by srIκBα overexpression.Through monitoring tumor growth using in vivo bioluminescence imaging system in orthotopic xenograft models,overexpression of TRIM22 in GBM cells was shown to accelerate the growth significantly in vivo and shorten median survival time of tumor-bearing mice compared to Flag-EV groups,but abolished by srIκBα overexpression.Conclusion1.TRIM22 is a critical oncogene that helps maintain high transcriptional activity of NF-κB in GBM cells;2.TRIM22 preferentially expressed in HGG,positively correlated with several clinicopathological factors;3.TRIM22 enhanced the K63-linked polyubiquitination level of IKKγ and K48-linked polyubiquitination level of IκBα through protein-protein interactions,in which activating NF-κB to promote GBM progression;... |
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