Pathogenesis Study Of LncRNA Hypoxia Regulation And Copy Number Variation In Glioma Malignant Progression

BackgroundHuman glioma is the most common malignant tumor in the central nervous system,accounting for about 80%of primary intracranial malignant tumors.Glioblastoma(GBM)is the most malignant type,accounting for more than half of all gliomas.Gliomas can be classified into 3 subtypes based on the gene expression signatures,including Proneural(PN)subtype,Classical(CL)subtype,and Mesenchymal(MES)subtype.Among these 3 subtypes,MES gliomas are highly malignant due to the features of invasive growth and resistance to radiotherapy and chemotherapy.Currently,the conventional treatment is maximum tumor resection based on functional area preservation,combined with temozolomide,radiotherapy,and standard adjuvant treatment.However,glioma patients have a high postoperative recurrence rate,short overall survival,and poor prognosis.The 5-year survival rate for GBM patients is only 5%.There are two main reasons for the situation.First,glioma,especially the MES subtype,is highly invasive.Tumor cells spread into the parenchyma of the surrounding brain,leaving great difficulties to surgical resection and postoperative radiotherapy.Secondly,glioma cells are resistant to most of the existing therapies.As tumor cells have gradually developed the resistance to various stresses and injuries through genomic evolution such as mutation or copy number variation(CNV),it is difficult for current radiotherapy,chemotherapy,and other adjuvant therapies to achieve the expected outcomes.Therefore,solving these two problems is of great significance to overcome glioma.Glioma invasive growth refers to the infiltration of tumor cells into the surrounding normal brain tissue,and there is no explicit boundary between tumor and normal tissue.Glioma cells spread away from tumor bulk to distant places through the intercellular space of the brain parenchyma,so the actual ranges of tumors always exceed what they are shown from the MRI by 1-2 cm,especially for the highly invasive MES subtype,which is characterized by the high level of VIM,CD44,CDH2(N-cadherin),and MMPs et al.Hypoxia is one of the key factors in the MES transition of glioma cells,and hypoxia-inducible factors(HIFs)are the critical transcriptional regulatory factors for tumor cells to adapt to the hypoxic microenvironment.They bind to the hypoxia response element(HRE)in the promoter region and regulate the expression of many genes involved with angiogenesis,migration,invasion,proliferation,apoptosis,metabolism,and many other biological activities.It has been found that genes such as ZEB1 and HIF1-AS2 are involved in hypoxia-induced MES transition of glioma,but little progress was made in clinical translation.Therefore,making further steps to reveal the key mechanisms involved in the MES transition of glioma cells under hypoxia is particularly important for improving glioma treatment strategies.DNA replication stress refers to the proliferating cells.When their genomes are exposed to various endogenous or exogenous stress during the replication process,the DNA replication forks are likely to run into stagnation.The stalled replication forks will further lead to DNA single-strand(ssDNA)accumulation or double-strand break(DSB)due to their unstable structure.The cells with intact DNA repairing function can resume the DNA replication process from the interrupted site after fixing the disadvantages or completing the DNA repair process.Nevertheless,for most tumor cells,their DNA repairing function is defective due to the mutation of DNA repair or checkpoint related genes,especially the mutations in the ATR-CHK1 pathway and ATM-CHK2 pathway,which lead to malfunction when DNA damage occurs,resulting in cell senescence or cell death.Scientists have invented radiotherapy and designed various chemotherapeutic drugs based on this feature.However,the effects of these treatments are always better at the early stage,and gradually reduce over time.It is due to the evolution of tumor genomes such as CNV.CNV is a common genomic structural variation that results in tumorigenesis.It is caused by the gain or loss of DNA fragments in a certain region of chromosomes,which leads to gene expression changes of corresponding region.For example,the gain of oncogenes or the loss of tumor suppressor genes will lead to aberrant cell proliferation or other malignant biological behaviors,and finally,form tumors or make them more malignant.The most common CNVs in GBM are chromosome 7 gain and chromosome 10 loss.Exploring the function of genes located in the high-frequency CNV regions is essential for decoding the secrets in tumor formation and progression,which is essential for the research and development of new precision treatments.At the same time,the discovery of long noncoding RNAs(lncRNAs)expands the space for tumor genomic research and brings new hopes for conquering tumors.Long noncoding RNAs(lncRNAs)are RNAs more than 200 nt in length without coding function and are poorly conserved among species.LncRNAs can bind with DNAs,RNAs,and proteins to affect transcription and translation processes.They can also participate in a wide range of biological activities by changing the stability and binding capacity of molecules.Recently,studies have found that some lncRNAs have protein-coding abilities,and the small peptides they encoded were proved to have important functions.These findings further expand the research landscape of lncRNAs.The role of lncRNA malfunction on tumor development is currently a hotspot in cancer research.The aberrant expression pattern of lncRNAs caused by transcriptional regulation or tumor genomic changes also significantly affects the malignant behavior of GBMs.This promising field still needs to be further explored.In this project,we used two ways to study the aberrant expression of lncRNAs and related malignant biological behaviors of glioma based on the online databases.First,given the characteristics of hypoxia promoting the MES transition of glioma cells,we screened and found that lncRNA PDIA3P1 was highly expressed in hypoxic glioma cells and facilitated their MES transition.Second,based on the characteristic that most primary GBMs have chromosome 7 copy number gains(CNGs),we found a new lncRNA AC093673.1 which located at 7q34,and verified that the upregulation of AC093673.1 reduced the DNA replication stress of GBM cells and contributed to the tumorigenesis and progression of primary GBM.The discovery of LncRNA PDIA3P1 and AC093673.1 provides a basis for accurate diagnosis and treatment of GBM patients,which has important translational significance.Part I:Mechanism study of hypoxia-induced lncRNA PDIA3P1 promoting mesenchymal transition in glioblastomaObjectives(1)Identify the upregulated lncRNAs in glioma under hypoxic condition(PDIA3P1).(2)Explore the relationship between PDIA3P1 and hypoxia-related malignant behaviors of glioma according to data analysis and experimental verification.(3)Investigate the mechanism of hypoxic regulation on PDIA3P1.Methods(1)Sequencing data acquisition from public platforms and enrichment analysis of DEGsThe RNA sequencing data of U87MG cell lines maintained under normoxia and hypoxia conditions were obtained from the GEO database(GSE45301).The sequencing and clinical data of glioma samples were obtained from the TCGA database and CGGA database.The PDIA3P1 expression cut-off value is taken from the expression level of the median sample.DESeq2 package from R language was used to calculate the differentially expressed genes(DEGs)(|log2FC|?2,P<0.05),and the online enrichment analysis software DAVID and GSEA were used for enrichment analysis.(2)Cell culture,transfection,and construction of PDIA3P1 stable knockdown/overexpressing glioma cellsHypoxic glioma cells were maintained in a hypoxic incubator(1%O2,5%CO2,37?).MicroRNA mimics,microRNA inhibitors,siRNAs,corresponding control sequences,pGL3-PDIA3P1 wild-type and mutant-type luciferase reporter plasmids,pGL3-RELA 3'UTR wild-type and mutant-type luciferase reporter plasmids,PDIA3P1 promoter and corresponding truncated luciferase reporter plasmids were transfected with lipo3000.Plasmids containing the full-length PDIA3P1 were constructed into pLVX-IRES-Puro vectors for lentivirus packaging.Then the lentivirus overexpressing PDIA3P1 was transfected into U87MG and A172 cells.RT-qPCR assay confirmed the efficiency.(3)Detection of RNA and protein expression levelsAfter the glioma cells were processed,total RNA or total protein was extracted,RT-qPCR and western-blot were used to detect the expression levels of mRNAs,microRNAs,and proteins in corresponding cells.Xenograft brain samples were embedded into paraffin and sliced into 5?m sections.The expression level of related proteins was detected by immunohistochemistry(IHC).(4)Migration and invasion ability assayTranswell assay was performed to evaluate cell migration ability.The matrigel-coated transwell chamber and 3D spheroid invasion assay was performed to determine the invasion ability of glioma cells in vitro.(5)Fluorescence in situ hybridization(FISH)After glioma cells were fixed with paraformaldehyde,FISH experiments were performed using PDIA3P1 probe or miR-124-3p probe to determine the RNA distribution and expression.(6)Biotin-labeled RNA pulldown experimentThe biotin-labeled miR-124-3p and control sequences were transfected into U87MG and U251 cells.After cell lysis,the magnetic beads were used to isolate and purify the products.RT-qPCR was used to evaluate the level of PDIA3P1 and RELA in miR-124-3p binding RNAs.(7)Chromatin immunoprecipitation(ChIP)According to the predicted HRE in the promoter region of PDIA3P1 and the luciferase reporter assay results,ChIP experiment was used to verify the direct functional HRE in the promoter region of PDIA3P1.(8)In situ xenograft nude mice modelA stereotactic syringe was used to inject the control and PDIA3P1 overexpressing luciferase-labeled U87MG cells into the brain of nude mice.The tumor viability was monitored by the Spectrum In Vivo Imaging System.The samples were embedded in paraffin for HE staining or IHC assay.Results(1)Hypoxia upregulates PDIA3P1,which is related to the MES subtype andpatient prognosisAccording to the GEO database,we found that the expression of PDIA3P1 was increased in hypoxic U87MG cells.Combined with the TCGA and CGGA databases,we found that the expression of PDIA3P1 in glioma samples was significantly higher compared with normal brain tissues,especially in highly-invasive MES gliomas.Moreover,we found that the overall survival(OS)of glioma patients with high PDIA3P1 expression was significantly shortened based on the clinical data.(2)PDIA3P1 promotes the migration and invasion of glioma cells in vivo and in vitroEnrichment analysis revealed that PDIA3P1 was significantly related to features such as MES transition,hypoxia,and extracellular matrix disassembly.Transwell,3D invasion assay,and in vivo experiment confirmed that PDIA3P1 could promote tumor cell migration and invasion.Western-blot assay detecting MES markers like Vimentin,CD44 et al.verified that PDIA3P1 promotes MES transition of glioma cells.(3)PDAI3P1 acts as ceRNA to sponge miR-124-3p in glioma cellsFISH and subcellular fraction RT-qPCR assay confirmed that PDIA3P1 is mainly located in the cytoplasm of glioma cells.Data analysis and RT-qPCR assays found that miR-124-3p was negatively correlated with PDIA3P1.Luciferase reporter assay confirmed that miR-124-3p suppressed the expression of PDIA3P1 reporter gene,and RNA pulldown assay finally demonstrated that miR-124-3p and PDIA3P1 had a direct interaction.(4)MiR-124-3p inhibits the migration and invasion abilities of glioma cells via targeting RELAMir-124-3p transfection changed the morphology of glioma cells.Transwell assay confirmed that overexpression of miR-124-3p impeded the migration and invasion of glioma cells.Western-blot testing MES markers like Vimentin,CD44 et al.verified that miR-124-3p inhibited the MES transition of glioma cells,while knocking down miR-124-3p had opposite effects.Online target prediction,RT-qPCR,and luciferase reporter gene assays confirmed that RELA was a direct target of miR-124-3p.(5)PDIA3P1 promotes glioma migration,invasion,and MES transition through activating NF-?B pathwayEnrichment analysis found that PDIA3P1 was closely related to the NF-?B pathway.Western-blot and IHC assays confirmed that PDIA3P1 upregulated the expression of p65 protein and activated the NF-?B pathway.Transwell and 3D spheroid invasion assays verified that RELA knockdown inhibited the migration and invasion of glioma cells.Western-blot confirmed that RELA knockdown hampered the MES transition of glioma cells.(6)HIF-1 heterodimer binds to the promoter region of PDIA3P1 and promotes its expressionData analysis and RT-qPCR assays demonstrated that HIF1A RNA expression was positively correlated with PDIA3P1 but negatively correlated with miR-124-3p.Western-blot found that overexpression of HIF-1? promoted the MES transition of glioma cells.Luciferase reporter assay using the truncated promotor plasmids and TSS mutant plasmids discovered two functional HRE in the promoter region of PDIA3P1.ChIP experiment confirmed the direct interaction between HIF-1 heterodimer and the functional HRE sites.Conclusion(1)Hypoxia-induced HIF-1 heterodimer upregulates PDIA3P1,and activates NF-?B pathway through PDIA3P1/miR-124-3p/RELA axis,promoting the MES transition of gliomas.(2)PDIA3P1 can be used as a diagnostic marker of MES gliomas,and is also a promising target for patient prognosis and targeted therapy.Part ?:Mechanism study of lncRNA AC093673.1 copy number gain relieves DNA replication stress in glioblastomaObjectives(1)Identify the upregulated lncRNAs in GBMs with chr 7 copy number gains.(AC093673.1).(2)According to the result of transcriptome sequencing,explore the role of AC093673.1 in the malignant progression of GBMs.(3)Explore the mechanism of AC093673.1 on GBM malignant progression.Methods(1)Sequencing data acquisition from public platforms and enrichment analysis of DEGsThe RNA-seq data DNA copy number variation data and clinical attributes of GBM samples were acquired from the TCGA database.DESeq2 package from R language was used to calculate the differentially expressed genes(DEGs)(|log2FC|?2,P<0.05).(2)Cell culture,transfection,and PDIA3P1 overexpressing GBM cell line constructionsi-AC093673.1 and control sequence,AC093673.1-WT,AC093673.1-ORF,5'UTR+ORF,and corresponding transcription start site(TSS)mutant plasmids,EGFP-tagged plasmids,and HA-tagged plasmids were transfected using lipo3000.Sh-AC093673.1 and control lentivirus,AC093673.1 overexpressing and control lentivirus were used to construct AC093673.1 stable knockdown/overexpressing GBM cells.RT-qPCR was used to verify transfection efficiency.(3)AC093673.1 knockdown in LN229 cells and transcriptome sequencingThree independent si-AC093673.1 sequences were used to knock down AC093673.1 in LN229 cells.Cells were collected,and total RNA was extracted after 48 hours.RNA-seq and data analysis were performed by Novogene.(4)Cell proliferation assaysAfter GBM cells were processed,cell viability was detected by CCK8 assay,and the DNA replication ability was evaluated by EdU assay.Cells were treated with propidium iodide(PI),and flow cytometry(FACS)was used to determine the cell cycle distribution.(5)DNA damage and cell apoptosis evaluationAfter GBM cells were processed,anti-ssRNA and anti-yH2AX were used for immunofluorescence(IF)staining to detect the DNA damage caused by replication stress or DSB.Alkaline comet assay was used to detect the total DNA damage in GBM cells.For apoptosis assay,Annexin V-FITC and PI-PE double staining were performed,and the ratio of Annexin V+cells was determined by flow cytometry.(6)FISH,RNA,and protein level detectionFISH assay was performed to determine the location of the lncRNAs.RT-qPCR,western-blot,and IF were used to determine the expression level of RNAs and proteins.(7)Evaluation of AC093673.1 coding capacityAC093673.1-ORF+EGFP,5'UTR+OR F+EGFP,ORF+HA,HA-Con,and corresponding TSS mutant plasmids were transfected into HEK-293T cells.The EGFP fluorescence and HA-tag expression were used to determine the protein-coding capacity after plasmids transfection.(8)In situ xenograft nude mice modelStereotactic syringe was used to inject the control and AC093673.1 knockdown luciferase-labeled GSC267 cells into the brain of nude mice.The tumor viability was monitored by the Spectrum In Vivo Imaging System.Results(1)AC093673.1 is upregulated in GBM samples with chr 7 CNGs,which correlates with poor prognosis of GBM patientsBy analyzing the TCGA-GBM database,we found that AC093673.1 was significantly upregulated in GBMs with chr 7 CNGs.Further study found that AC093673.1 was located at chromosome 7q34,high AC093673.1 expression or CNGs in the corresponding region was correlated with poor outcomes for GBM patients.(2)AC093673.1 is enriched in the cytoplasm and has protein-coding abilityFISH experiment and subcellular components RT-qPCR confirmed that AC093673.1 is mainly located in the cytoplasm.Transfection of AC093673.1-ORF plasmids with wild-type or mutant TSS,and EGFP or HA-tag confirmed that AC093673.1-ORF has coding ability,and the peptides it encoded were mainly located in the nucleus of GBM cells.(3)AC093673.1 knockdown inhibits GBM cell proliferationRNA-seq results suggest that AC093673.1 is related to cell cycle checkpoint and DNA replication.CCK8 experiment confirmed that the cell viability was significantly reduced,and EdU assay found that the DNA synthesis speed was decreased after AC093673.1 knockdown in GBM cells.Cell cycle determination using FACS found that the cells exhibited G1/S arrest after AC093673.1 knockdown,while overexpression of AC093673.1 ORF promoted G1/S transition of GBM cells.(4)AC093673.1 knockdown aggravates DNA damage and apoptosis of GBM cellsRNA-seq results suggested that AC093673.1 was related to DNA damage repair.IF assay detecting y-H2AX and ssDNA,in addition to comet assay confirmed the increased DNA damage level after AC093673.1 knockdown.FACS apoptosis assay demonstrated that the proportion of apoptotic cells in AC093673.1 knockdown GBM cells was significantly increased regardless of whether cells received radiotherapy.Western-blot assay found that AC093673.1 knockdown in GBM cells significantly impeded the DNA repair process after radiotherapy.In vivo experiments also confirmed that AC093673.1 knockdown inhibited the viability of GSC267 cells in the brain of nude mice.(5)AC093673.1 ameliorates DNA replication stress through ATR-CHK1 pathway in GBMsEnrichment analysis found that AC093673.1 was significantly related to DNA replication stress and damage repair checkpoint ATR-CHK1.Western-blot confirmed that AC093673.1 knockdown in GBM cells significantly inhibited th e ATR-CHK1 checkpoint.ssDNA staining,comet assay and FACS apoptosis assay confirmed that overexpression of AC093673.1 in GBM cells endowed them with better tolerance to HU-induced DNA replication stress.Western-blot assay confirmed that overexpression of AC093673.1 upregulated the activity of ATR-CHK1 pathway,which was a crucial factor that promoted GBM malignant progression.Conclusion(1)LncRNA AC093673.1 is upregulated in GBMs with chr 7 CNGs,which encodes short peptides and activates ATR-CHK1 pathway,ensuring the rapid proliferation of tumor cells and reducing the DNA damage and cell death caused by DNA replication stress.(2)AC093673.1 is an important indicator for the viability of GBMs,and it is also a promising therapeutic target for GBM treatment.