The Role And Mechanism Of Endoplasmic Reticulum Stress Mediating DERL3-HNRNPA2B1 Axis In Regulating Malignant Progression Of Glioma

Objective: Glioma is the most common primary intracranial malignant tumor.Due to its high invasiveness and malignant proliferative ability,it leads to high recurrence rate and mortality rate,especially in glioblastoma(GBM).Even with the standardized treatment strategy,that is,maximal surgical resection combined with chemotherapy or radiotherapy,the prognosis of glioma is still very poor.At present,glioma is treated by maximal surgical resection combined with radiotherapy and chemotherapy,but the treatment effect is poor.In recent years,more and more studies have begun to focus on the crosstalk between malignant tumor cells and their microenvironment.Various extracellular signals or treatments often make tumor cells into a high stress state,such as mitochondrial stress or endoplasmic reticulum stress activation.This kind of stress can lead to apoptosis at first,but will finally lead to treatment resistance,largely because the tumor cells have the ability to adapt to this kind of stress or even take advantage of this stress to activate specific cellular signals to promote proliferation.Identification of stress-related targets and regulatory mechanisms in glioma is of great significance for understanding the adaptation of glioma cells under stress and developing relevant targeted therapies.In this study,we established an individual ER-stress risk score from the glioma public database and found that the risk score was closely related to the poor prognosis,malignant molecular subtypes and biological functions of glioma.Subsequently,we screened and identified the core gene DERL3 that could represent this risk score,and confirmed that DERL3,as an endoplasmic reticulum stress effector,participating in the regulation of malignant proliferation and invasion of glioma cells using in vitro and in vivo experiments.We also elucidate the deep molecular mechanism of this regulatory effect dependent on the activation of NF-κB pathway by DERL3 binding HNRNPA2B1 and maintaining its stability.Methods:(1)Database information and glioma tissue specimens: Relevant bioinformatics analysis data were obtained from Chinese Glioma Genome Atlas(CGGA),The Cancer Genome Atlas(TCGA).CGGA was selected as the internal discover database.TCGA was selected as external validation databases.Glioma tissue specimens were obtained from the First Hospital of China Medical University with the informed consent from patients and their families.Relevant experimental plans were reviewed and approved by the Ethics Committee of the First Hospital of China Medical University.(2)Bioinformatics analysis methods: Gene ontology(GO)analysis and KEGG analysis were used to annotate the biological function and molecular pathway of genes that differed between different groups;Gene set enrichment analysis(GSEA)software was used to analyze the pathway activity alteration between different groups.Univariate COX regression analysis was used to analyze the survival of specific gene and multivariate COX regression analysis was used to explore the independent prognostic factors after univariate survival analysis.ROC analysis was performed for independent prognostic factors in multivariate COX regression analysis to further verify the ability of the model to predict survival prognosis.(3)Cell line research model: Human glioma cell lines U87,LN229 and U251 were purchased from Shanghai Cell Bank,Chinese Academy of Sciences.Human glioma cell line was purchased from American Type Culture Collection.Normal human astrocyte was a gift of professor Jiang from Beijing Neurosurgical Institute.GSC21 and GSC40 were patient derived primary cell lines extracted by our laboratory team.The extraction protocol was approved by the Ethics Committee of the First Hospital of China Medical University.(4)Molecular biology experiments: Quantitative real time polymerase chain reaction(q-PCR),Western Blot(WB),immunohistochemistry(IHC)and immunofluorescence(IF)were used to verify the molecular expression;Immunoprecipitation(IP)and liquid chromatography-mass spectrometry(LC/MS)were used to identify potential receptor proteins binding to DERL3.MTS was used to detect the proliferation of glioma cells.Transwell assay was used to detect the invasive and migrating ability of glioma cells.Glioma intracranial xenograft in Balb/c nude mice was used for in vivo validation of relating phenotypes.(5)Statistical analysis: Student’s T test,One way ANOVA and Chi-square test were used to detect whether there were differences among variables in different groups.Kaplan-Meier curves were drawn and Log-rank test was performed to compare the differences in survival status among patients in different groups.COX regression analysis was used to determine the factors affecting the survival and prognosis of patients.Correlation analysis was done using Pearson test.Graph Pad Prism 7,SPSS and R language were used to complete the above analysis.The two-tailed P value less than0.05 was defined as statistically significant.Results:Part Ⅰ: Description of clinical value of endoplasmic reticulum stress state in glioma and identification of DERL3 as core endoplasmic reticulum stress geneFirst,we summarized the endoplasmic reticulum stress related gene sets.Based on CGGA and TCGA glioma dataset,we performed univariate COX analysis and screened seven genes with P < 0.05.Based on the seven genes,we built individualized glioma endoplasmic reticulum stress related risk score.We found that the risk score was related with patient’s pathological status.The score in IDH1 wild type patients was higher than that in IDH1 mutant type patients and the score in mesenchymal subtype patients was higher than that in other molecular subtypes.Survival analysis suggest high endoplasmic reticulum stress risk score was associated with shorter survival time.We further used dimension reduction analysis to identify the core of endoplasmic reticulum stress related genes,DERL3.DERL3 could be used to represent ER-stress risk score.We found that the expression of DERL3 was related with patient’s pathological status.The expression of DERL3 in IDH1 wild type patients was higher than that in IDH1 mutant type patients and the expression in mesenchymal subtype patients was higher than that in other molecular subtypes.Survival analysis suggest high DERL3 expression was associated with shorter survival time.These results suggest that DERL3 may be a potential marker gene and regulatory target related to ER stress in gliomas.Subsequently,the expression pattern of DERL3 in glioma patients and cell lines was analyzed by immunohistochemistry,Western Blot and q PCR,which confirmed that DERL3 was positively correlated with the malignant degree of glioma,laying a foundation for further research on whether DERL3 plays a regulatory role in the malignant progression of glioma.Part Ⅱ: The mechanism of DERL3 on malignant progression of glioma cells which is regulated by ER-stressIn the first part,we described the clinical value of ER stress-related risk score using glioma database,and identified the relevant core molecule DERL3.Based on the preliminary experimental results,we selected U87 and LN229 glioma cell lines for subsequent molecular biological experiments.First,we constructed DERL3 lentivirus knockdown and plasmid vector overexpression cell lines respectively.Subsequent functional phenotype test results showed that DERL3 knockdown significantly inhibited proliferation,invasion and intracranial tumor-forming ability of glioma cells.On the contrary,the overexpression of DERL3 promotes the proliferation and invasion of glioma cells,suggesting the important regulatory role of DERL3 in the tumorigenesis and development of glioma malignancies.In order to explore the regulatory relationship between DERL3 and ER-stress state,we carried out a series of related molecular biology experiments.First,the intervention of DERL3 expression did not change the ER-stress status of glioma cells.On the contrary,when the ER stress status of glioma cells was activated or inhibited,the expression of DERL3 was consistent with the expression of ER-stress related markers,indicating that the expression of DERL3 was regulated by ER-stress status of glioma cells,and it could also be used as a new marker of ER stress activation.At the same time,we found that the malignant behavior of glioma cells was enhanced after ER-stress activation,and DERL3 knockdown significantly rescued the malignant effect caused by ER stress activation,suggesting that ER-stress promoted the malignant progression of glioma cells partly dependent on DERL3.These results suggest the important mediating role of DERL3 in ER-stress in promoting glioma malignant progression,and it can be used as a good regulatory target of glioma stress.Part Ⅲ: The mechanism of DERL3-HNRNPA2B1 signaling axis affecting the malignant progression of glioma cells by activating the NF-κB pathwayIn order to further explore the molecular mechanism of DERL3 promoting the malignant progression of glioma,we firstly screened differential genes according to the expression level of DERL3 using glioma database.The results of enrichment analysis suggested that the high expression of DERL3 was related to the activation of NF-κB pathway.Subsequently,using previously established DERL3 knockdown and overexpression cell line we confirmed that intervention of DERL3 expression could either inhibit or activate the NF-κB pathway,respectively.We further confirmed that the role of DERL3 in promoting the malignant progression of glioma cells depends on the activation of NFKB pathway with key rescue experiments.In order to further elucidate the molecular mechanism of DERL3 activation of NF-κB pathway,we conducted DERL3 immunoprecipitation combined WB test for mass spectrometry analysis.HNRNPA2B1 was selected as poteintial genes.It has been reported that HNRNPA2B1 has a regulatory effect on the activation of NF-κB pathway.Subsequently,the Co-IP experiment was used to reconfirm the binding relationship between DERL3 and HNRNPA2B1,and it was found that the combination of DERL3 increased the protein stability of HNRNPA2B1 and reduced the degradation of HNRNPA2B1 through proteasome pathway,thus clarifying the post-transcriptional regulation mechanism of DERL3 on HNRNPA2B1.Finally,through a series of rescue experiments,we confirmed that DERL3 activated the NF-κB pathway of glioma cells through the mediation of HNRNPA2B1 to promote the malignant progression of glioma.Conclusions:Part Ⅰ: Endoplasmic reticulum stress risk score established by us can be used as an important marker to judge the survival status and adverse molecular events of GBM patients;DERL3 can be used as a reliable core representative molecule of er stress and marker of glioma malignancies.Part Ⅱ: DERL3 has a regulatory function in glioma cells,and intervention of DERL3 can significantly reduce the malignant progression of glioma.DERL3 can be used as a marker of ER-stress activation,which mediates the promoting effect of ER stress on malignant behavior of glioma cells.Part Ⅲ: DERL3 specifically binds HNRNPA2B1 molecules and maintains HNRNPA2B1 expression by inhibiting its proteasome pathway degradation.DERL3-HNRNPA2B1 signaling axis promotes the malignant progression of glioma through the mediation of NF-κB signaling pathway.Targeting DERL3-HNRNPA2B1-NF-κB signal axis will provide new treatment strategies of glioma,which has important scientific value and clinical significance.