GDNF Promotes The Proliferation Of Glioblastoma Cells Through NRP1-?-catenin-CXCL1

Glioblastoma is the most common primary tumor in the central nervous system,with rapid growth,high degree of malignancy and poor prognosis.It was found that glial cell line-derived neurotrophic factor(GDNF)was highly expressed in glioma and could promote the proliferation of glioma cells.GDNF is a member of the transforming growth factor-beta(TGF-?)superfamily,which can promote the proliferation of glioma cells by binding to membrane receptors and activating the intracellular signaling pathways,but the specific mechanism of promoting the proliferation of glioma cells of GDNF is unclear.It is known that membrane receptors of GDNF are diverse and that different membrane receptors may be present in different cells,and then the intracellular signal transduction pathways and the final biological effects are also different.In this study,rat glioblastoma cell line C6 cells were chosen to explore the key membrane receptor,intracellular signaling pathways and proliferation-related target gene of GDNF in promoting the proliferation of glioma cells,hoping to find potential biological targets that can block glioma cell proliferation,thereby improving the prognosis of glioma patients.Object To explore the possible membrane receptors of GDNF,related intracellular signaling pathways,and proliferation-related target genes in the process of proliferation of C6 rat glioma cells induced by exogenous GDNF.Methods C6 cells were cloned from N-nitrosomethyl-induced rat glioblastoma cells by Benda et al.,and were cultured in vitro and animal passages for many times.It is an experimental widely used glioma cell line.In this study,C6 glioma cells were purchased from the Chinese Academy of Sciences cell / stem cell bank.1.Logarithmic growth period C6 cells and normal primary astrocytes(AST)cells,normally cultured,were selected to study.Flow cytometry was used to determine the serum starvation conditions for C6 glioma cell G0 / G1 phase synchronization;CCK8 assay was taken to find the best concentration and time point of exogenous exogenous GDNF to promote C6 glioma cell,and the results were verified by flow cytometry and Ed U staining;2.C6 cells and primary astrocytes were collected to extracting membrane proteins;GST and GST-GDNF fusion proteins were used as bait to carry out GST pull-down experiments on membrane proteins,and high performance liquid chromatography tandem mass spectrometry(LC-MS / MS)and gene ontology(GO)were used in further to screen proteins that might bind to GDNF;3.The neuroplin-1(NRP1)was screened for further study;immunofluorescence staining was selected to observe the expression of NRP1 protein in C6 cells;the expression of NRP1 m RNA in brain gliomas and normal brain microarray was analyzed by Oncomine platform;laser confocal technique was taken to detect the effect of exogenous GDNF on the distribution of NRP1 in C6 glioma cells;4.The protein docking between GDNF and NRP1 was taken by ZDOCK server,and the best docking mode between the two was extracted;further analysis of hydrogen bonds and electrostatic interactions between GDNF and NRP1 was done by Py MOL software;immunoprecipitation technique was taken to determine whether NRP1 and GDNF can be combined or not;RT-PCR and WB experiments were used to investigate theeffect of exogenous GDNF on NRP1 gene and protein level;5.The RNA interference lentiviral vector of rat NRP1 gene was constructed,and the C6 cells were infected with it;CCK8 assay was used to detect the effect of exogenous GDNF on the proliferation of C6 cells treated with anti-NRP1 antibody and NRP1-RNAi lentivirus,respectively;6.The effects of exogenous GDNF on the expression of protein and m RNA of NRP1 were measured by Western Blot and polymerase chain reaction(PCR);the data of glioblastoma were downloaded from c Bioportal,and the effects of NRP1 overexpression on survival and prognosis of patients were analyzed by two-sample rank sum test,Cox regression model and Kaplan-Meier survival curve;7.After the cell cycle synchronization of C6 cells was initiated by serum starvation,exogenous GDNF was treated and the cytoplasm and nuclear protein were extracted;the translation of ?-catenin from cytoplasm to nucleus was observed by WB and immunofluorescence staining;8.The above-mentioned extracted cytoplasm and nuclear protein were subjected to immunoprecipitation(IP)with ?-catenin antibody,and then the enrichment of the protein obtained by IP was analyzed by mass spectrometry to achieve the differential expression of protein interacted with ?-catenin under the condition with/without GDNF;and then using DAVID software for GO(Gene Ontology)ontology and KEGG(Kyoto Encyclopedia of Genes and Genomes)pathway analysis,the protein-protein interactions(PPI)network analysis was performed by STRING and Cytoscape software(analysis target: differentially expressed genes and GDNF,NRP1 and CTNNB1);9.Rac1 was chosen as the research object,and the immunoprecipitation experiments between Rac1 and ?-catenin were carried out;10.After the cell cycle synchronization of C6 cells was initiated by serum starvation,total RNA was extracted by exogenous addition of GDNF on the different time of 0,0.5,1 or 24 h;the m RNA transcriptome sequencing was taken to find out the differential expression genes in exogenous GDNF 0.5,1 or 24 h group compared with 0 h group;bioinformatics technology was used to screen proliferation related genes ofGDNF,and the results were verified by RT-PCR;11.CXCL1 was chosen as the research object,and to investigate the effect of exogenous GDNF on the activity of CXCL1 promoter,the luciferase reporter assay was carried out;the bioinformatics method was used to explore the co-transcription factor of beta-catenin,which was capable of initiating CXCL1 transcriptionResults 1.Exogenous GDNF can promote the proliferation of C6 glioma cells,the best concentration is 40 ng / ml and the best time point is 48 h;2.Membrane protein was extracted and verified by Na / K ATPase,which is an internal reference of membrane protein;GST immunoprecipitation,Mass spectrometry and GO ontology analysis were used to screen membrane proteins that bind to GDNF,the results showed that compared with rat primary astrocytes,the specific membrane proteins that bind to GDNF on C6 glioma cells are attractin(ATRN),neuropilin-1(NRP1)and neurofilament protein 2(Neuropilin-2,NRP2);the Oncomine platform was used to analyze the m RNA expression between brain GBM and normal brain tissue,the data of brain GBM comes from the Cancer Genome Atlas(TCGA)data set,and the results showed that the expression of NRP1,NRP2 and integrin beta-1(ITGB1)was significantly increased,while the glial cell line-derived neurotrophic factor receptor ?1(GDNF family receptor alpha 1,GFR?1),receptor tyrosine kinase(RET),neuronal cell adhesion molecule(NCAM),cadherin-2(CDH2),poly-ligand proteoglycans(syndecan-3,SDC3)and ATRN(CDT2)had no significant difference;3.The neuroplin-1(NRP1)was chosen for further study;immunofluorescence staining pointed that the expression of NRP1 in C6 cells was significantly higher than that in normal astrocytes;laser confocal microscopy gave the result that exogenous administration of GDNF could induce more NRP1 onto the C6 glioma cell membrane;4.The protein docking between GDNF and NRP1 was performed by the ZDOCK server and analyzed by Py MOL software,the results showed that both could interact with each other by hydrogen bonding and electrostatic stabilization,and among them,the NO.201 residues of GDNF could form four hydrogen bonds with NRP1residues;immunoprecipitation techniques also demonstrate the ability of binding between NRP1 and GDNF;5.NRP1 interference lentivirus was constructed and infected into C6 cells successfully;in the case of NRP1 knockdown or using NRP1 neutralizing antibody,CCK8 test showed that the effect of exogenous GDNF on C6 cell proliferation was weakened;6.The results showed that the overall survival(OS)and disease-free survival(DFS)in high level of NRP1 m RNA group were significantly lower than that of other patients Western blot and PCR showed that exogenous GDNF induced increased expression level of protein and m RNA of NRP1 in C6 cells;the data of glioblastoma downloaded from c Bioportal were analyzed by two-sample rank sum test,Cox regression model and Kaplan-Meier survival curve;the results showed that the overall survival(OS)and disease-free survival(DFS)in high level of NRP1 m RNA group were significantly lower than that of other patients;7.Cytoplasm and nuclear protein were extracted and verified by WB with the internal reference of cytoplasm and nucleus;Western blot and immunofluorescence showed that ?-catenin transfered to the nucleus significantly after exogenous GDNF treatment,and levels of protein in the nucleus were elevated;8.IP and mass spectrometry results showed that the binding of Ras-related C3 botulinum toxin substrate 1(Rac1)to the ?-catenin was increased under the action of GDNF;bioinformatics analysis showed that the GO and KEGG of up-regulated protein were mainly enriched in mitochondrial-related entries;the results of constructing PPI networks through STRING and Cytoscape software showed that Rac1 may play a role in intracellular signaling from NRP1 to ?-catenin;9.Immunofluorescence and co-precipitation results showed that Rac1 could bind to ?-catenin in C6 cells;10.Total RNA was extracted after adding exogenous GDNF into C6 glioma cells;the results of m RNA sequencing showed that the expression of CXCL1 was up-regulated in exogenous GDNF at different time points,and the results were verified by RT-PCR;11.The results of luciferase reporter assay showed that exogenous GDNF enhanced theactivity of CXCL1 promoter.The bioinformatics method predicted that NF?B could initiate CXCL1 transcription as a co-transcription factor of ?-catenin.Conclusion The proliferation of GDNF glioma cells requires a specific transmembrane receptor to induce intracellular signaling.Our results suggest that transmembrane protein NRP-1 can bind to GDNF to initiate intracellular signaling and mediate the effect of promoting proliferation of GDNF.When GDNF-NRP1 is formed,Rac1-?-catenin is activated and undergoes nuclear transfer,which leads to an increase in the expression of proliferation-related gene CXCL1.In summary,we came up with a possible signaling pathway for GDNF-promoting glioma cell proliferation: GDNF-NRP-1-?-catenin-CXCL1.An in-depth study of this pathway is expected to provide a potential drug target for the treatment of glioma.