CRNDE Affects The Progression Of Glioma By Regulating ETS1/GPR17

Research background and purpose:Glioma is an aggressive primary brain tumor,which is featured as invasive growth and early metastasis.Glioblastoma multiforme(GBM)is the predominant subtype of all types of glioma with a survival rate generally less than 5 years.The high recurrence rate and low treatment efficiency of glioma lead to a poor prognosis.Although surgical resection,radiotherapy,chemotherapy,and targeted therapy have developed in recent decades,the treatment of glioma,especially GBM,is still not satisfactory.The development of glioma is complex,which is accomplished by multigene interaction and multimolecular regulation.It is vital to study the molecular mechanism of glioma for effective avenues and prognosis methods for glioma.lncRNAs have become a research hotspot due to their aberrant expression in various tissues,particularly in tumor tissues.Recent studies display that lncRNAs participate in varying biological processes in the human body,such as transcriptional activation of cells,intracellular transport,heat shock response,and genomic imprinting.lncRNAs are associated with various malignant tumors and exist in tumors as oncogenes and prooncogenic factors.There are also some reports on the impacts of lncRNAs on the proliferation and metastasis of glioma.Yan et al.have discovered that lncRNA Flv CR1-AS1 modulates the E2F2 level by competitive adsorption of mi R-4731-5p,further promoting proliferation and metastasis of glioma.A study by Li et al.proves that downregulation of LINC00174 can inhibit resistance to temozolomide in the glioma cells,the molecular mechanism of which is that LINC00174 can sponge mi R-138-5p to affect the expression of SOX9 and further improves the resistance to temozolomide and proliferation of glioma cells.In addition to serving as ce RNAs to further regulate the biological function of glioma through competitive adsorption of mi RNAs,lncRNAs can also directly regulate gene expression in combination with transcription factors to influence the development of glioma.Lv et al.found that lncRNA PVT1 can promote the proliferation and migration of glioma cells by downregulating the expression of UPF1.As a lncRNA,CRNDE exerts an essential role in a variety of cancers.Sun et al.reported that CRNDE promotes the chemosensitivity of medulloblastoma cells via modulating the mi R-29c-3p level.Bai et al.demonstrated that CRNDE as an oncogene in cervical cancer could promote the development of cervical cancer via increasing the CCNB1 level by competitively adsorbing mi R-183.These reports confirm the importance of CRNDE in cancer development.CRNDE functions in the development of glioma.Li et al.put forward that CRNDE as a ce RNA facilitates the progression of GBM via modulating the mi R-136-5P/Bcl-2/Wnt2 signal axis.Kiang et al.found that CRNDE functions in promoting glioma progression,while the promoting effect is regulated by the EGFR signal.However,the specific molecular mechanism of lncRNAs on glioma progression,especially the regulation of lncRNAs in combination with transcription factors on glioma progression,needs to be further explored.This study focuses on the effect of CRNDE on glioma and the specific molecular mechanism of CRNDE regulation of glioma,especially whether it can combine with related transcription factors to regulate downstream genes,thus affecting the occurrence and development of glioma.As well as the relationship between CRNDE and pathological features of clinical glioma patients.Research methods:Bioinformatics methods were used to study the relationship between CRNDE and glioma development through public databases LINCDISEASE,GSE50161,GEPIA,TCGA,and CGGA.q RT-PCR,Western blot,and non-radioactive in situ hybridization(FISH)were used to investigate the expression of CRNDE and the localization of suborganelles.To further explore the effects of CRNDE on the biological behavior of glioma cells and the possible molecular mechanism of CRNDE binding transcription factors to regulate downstream genes,cell counting kit 8(CCK-8),colony formation,apoptosis,and Transwell invasion detection were performed in vitro.Using public databases LINCDISEASE,GSE50161,Lnc MAP,and Jaspar,the downstream gene ETS1/GPR17 that may be regulated by CRNDE was predicted and demonstrated.Chromatin immunoprecipitation(CHIP)was used to analyze the relationship between ETS1 and GPR17.To construct glioma intracranial xenograft tumor model for in vivo experiment.Unpaired Student’s two-tailed T-EST test was used to detect the data difference between the two groups.The expression levels of CRNDE in gliomas of different WHO grades were compared by one-way ANOVA.Chi-squared test,using the high and low expression of CRNDE as truncation values,compared the distribution of gender,WHO grade,IDH status,and 1P / 19 Q co-deletion status between the low and high expression groups.Receiver operating characteristic(ROC)curves were used to test the prediction results of CRNDE.The Kaplan-Meier method was used to compare the overall survival(OS)of glioma patients between low and high CRNDE expression groups.Statistical analysis was performed using Graph Pad Prism 7(Graph Pad Software,Inc),R V3.4.1(https://www.r-proje ct.org/),and SPSS 20.0(SPSS Inc).All experiments were conducted independently for three or more times,and P <0.05 was considered statistically significant.Research result:Experiment 1: CRNDE expression was significantly up-regulated in glioma tumor samples.(1)To search for glioma-related DELncRNAs,we screened 142 glioma-related lncRNAs from the LINCDISEASE database.(2)limma differential analysis was performed on the GSE50161 chip in the GEO database,and 37 DELncRNAs were obtained.Taking the intersection of DELncRNAs and glioma-related LncRNAs,five glioma-related DELncRNAs were obtained,namely MEG3,CRNDE,XIST,Ag AP2-AS1,and LINC01116.(3)By analyzing the expression differences of these five lncRNAs in the normal samples of the GSE50161 chip and glioma tumor samples,it was found that the expression of CRNDE in tumor samples was significantly up-regulated,and the difference was the largest compared with normal samples.(4)Since there is no normal tissue data for GBM in the TCGA database,therefore,we compared the clinical expression data of CRNDE in the GEPIA database.The results showed that CRNDE was significantly up-regulated in LGG tumor samples,which was consistent with the trend of CRNDE in the GEO database.The levels of CRNDE in HEB cells and glioma cells T98 G,A172,SNB19,and U251 were detected by q RT-PCR.The results showed that CRNDE was significantly overexpressed in all 4 glioma cell lines compared with HEB cells.(5)For validation in multiple databases,in the TCGA and CGGA databases,we again analyzed the relationship between CRNDE and glioma grade.The results showed that CRNDE was positively correlated with glioma grade.Survival analysis and ROC results showed that CRNDE could significantly predict the prognosis of clinical glioma patients.(6)Combining the clinical data of TCGA and CGGA glioma patients,we found that the expression of CRNDE was significantly correlated with the clinicopathological features of glioma.Experiment 2: Overexpression of CRNDE regulates the proliferation,apoptosis,migration,and invasion of glioma cells.(1)In T98 G and U251 cells,exogenous overexpression of CRNDE significantly promoted cell viability and growth,and inhibited cell apoptosis.(2)FISH experiments and nuclear/plasma separation experiments confirmed that CRNDE was expressed in both the nucleus and cytoplasm of tumor tissues and cells,and the expression level in the nucleus was significantly higher than that in the cytoplasm.(3)In addition,we further examined the effect of CRNDE on the significantly enhanced migration and invasion of glioma cells after overexpression.After CRNDE overexpression,the expression levels of N-cadherin,Vimentin,MMP9,and MMP2 increased,while the expression level of E-cadherin decreased.Experiment 3: CRNDE promotes the expression of GPR17 by promoting the binding of ETS1 to the GPR17 promoter region.(1)To explore the downstream transcription factors of CRNDE and target m RNAs that regulate glioma cells.We used the lnc MAP database to predict the regulatory triplet lncRNA-TF-m RNA of CRNDE in glioma and obtained 30 downstream regulatory transcription factors.The crossover of these 30 transcription factors with prominent dem RNAs in GSE50161 resulted in four candidate transcription factors,ETS1,MYC,NR3C2,and FOXP2.By analyzing the expression differences of these four transcription factors in the GSE50161 chip between normal samples and glioma tumor samples,we found that the expression of ETS1 was significantly up-regulated in tumor samples,with the most significant difference compared with normal samples.In addition,we compared the m RNA expression data of ETS1 in the GEPIA database,and the results showed that ETS1 was significantly up-regulated in LGG tumor samples.At the same time,GPR17 was obtained by intersecting the delncRNA in GSE50161 with the m RNA in the lnc MAP triplet prediction result.The expression of GPR17 in the GSE50161 chip and GEPIA database clinical data showed that GPR17 was highly expressed in tumor samples.(2)q RTPCR detection showed that overexpression of exogenous CRNDE could promote the expression of GPR17 in glioma cells.To investigate whether ETS1 binds to the GPR17 promoter region to regulate GPR17 transcription,we used the Jaspar database to predict the binding sites of ETS1 and GPR17 promoter regions,and microarray and q RT-PCR assays were used to demonstrate that ETS1 can bind to GPR17.to the GPR17 promoter region.(3)The cells were divided into oe-NC + sh-NC group,oe-CRNDE + sh-NC group,and oe-CRNDE + sh-ETS1 group.The expression of GPR17 protein and m RNA in each group was detected respectively.We found that silencing ETS1 suppressed the promoting effect of GPR17 overexpression in both glioma cells.These results demonstrate the interaction of CRNDE-ETS1-GPR17,suggesting that CRNDE may promote the expression of GPR17 by regulating ETS1.Experiment 4: CRNDE regulates GPR17 to modulate glioma tumor cell phenotype.(1)Reversion experiments were performed in T98 G and U251 cells.The results of the MTT assay showed that the cell activity was significantly enhanced after overexpression of CRNDE,and the promotion of CRNDE overexpression was significantly weakened after GPR17 inhibition.Apoptotic results are opposite to proliferative results.(2)In terms of cell migration and invasion,overexpression of CRNDE and inhibition of GPR17 inhibited the promoting effect of CRNDE overexpression on cells.The levels of invasion and migration-related proteins were detected by Western blot,and the results also showed that oe-CRNDE + sh-GPR17 reversed the ability to overexpress CRNDE to regulate protein expression alone.Experiment 5: CRNDE promotes the malignant progression of glioma in vivo.(1)U87 cells were transfected with sh-CENDE lentivirus containing a luciferase reporter gene.By establishing an intracranial glioma model of U87 cells,the role of CRNDE in promoting tumor malignant progression was further confirmed in vivo.(2)Kaplan-Meier analysis and immunohistochemistry of nude mice after tumor formation showed that knockdown of CRNDE would prolong the survival days of nude mice and suppress the proliferation and migration ability of gliomas,which is consistent with the conclusions drawn from in vitro cell experiments.Analysis conclusion:The study revealed that CRNDE promotes the development of glioma cells.CRNDE combined with ETS1 up-regulates the expression of GPR17,promotes the proliferation,migration,and invasion of glioma cells,and hinders cell apoptosis.