The Roles And Molecular Mechanisms Of The Ubiquitin Ligase RNF126 In Human Glioma Cell Proliferation

Glioma is the most common tumor in the central nervous system,accounting for about 44.69%of intracranial tumors,ranking first in the incidence of intracranial tumors.At present,the treatment of glioma is still very poor,generally with a 5 year survival rate is only about 5%,the world still no good treatment,the root causes of poor prognosis of glioma is a series of biological behavior of malignant tumior cells,including proliferation,on brain tissue invasion,chemotherapy drug resistance,especially the excessive proliferation of tumor cells,is an important factor leading to rapid recurrence of glioma after operation.In the treatment of glioma while taking radiotherapy and chemotherapy,surgery and other means,but the treatment effect is still not ideal,so in recent years research on molecular glioma therapy has become a hot topic in the treatment of glioma.However,the understanding of the pathogenesis and pathogenesis of glioma is still relatively weak,so it is of great significance to find out the pathogenesis of glioma and propose effective molecular targeted therapy.There are mainly two kinds of eukaryotic protein degradation pathway:a lysosomal pathway,mainly by endocytosis into the degradation of proteins in cells;the other is the non lysosomal pathway,mainly by protease particles in the body cell(proteasome)degradation of ubiquitination of intracellular protein.Ubiquitin is a protein composed of 76 amino acids,protein ubiquitination typically requires three enzyme has catalysis,including ubiquitin-activating enzyme(E1),ubiquitin-conjugating enzyme(E2)and ubiqxuitin ligase(E3),which controls the specific catalytic substrate.The ubiquitin proteasome pathway by ATP and ubiquitin dependent mechanism through ubiquitin activating enzyme E1 and the ubiquitin ligase E2 and the combination of multi step enzyme ubiquitin protein ligase E3 in the catalytic reaction,the specific amino acid residues with protein substrates on multiple ubiquitin units,complete protein substrate ubiquitination.Studies have shown that a large nunber of proteins can be transcribed and modified by ubiquitin.Ubiquitin is covalently bound to the lysine residue of the substrate protein via a three step enzymatic reaction.The ubiquitin is first activated by El(ubiquitin activating enzyme),and the activated ubiquitin is then delivered to the ubiquitin conjugating enzyme E2 and then transferred from ubiquitin to the lysine residue of the target protein via the ubiquitin ligase E3 E2.In general,the ubiquitination binding enzyme E2 determines the type of ubiquitination modification,whereas ubiquitination of E3 determines substrate specificity.At present,two E1 enzymes,50 E2 enzymes and 600 E3 enzymes have been identified from the human genome.The E3 family has now been found to have two main categories,including the majority of the RING-finger family and the HECT(homologous to E6AP C terminus)family.The E3 of the RING-finger family contains similar E2 binding domains,and bridges,as a bridge,activate the ubiquitin directly from E2 to target proteins,which in themselves do not interact with ubiquitin.The HECT family of E3 and E2 in combination,contains the catalytic domain of HECT,Cys and E2 conserved residues with ubiquitin thioester bond formation,E2 first ubiquitin passed to E3,then the E3 presenting to the substrate.With the discovery of more and more novel ubiquitin ligase,the understanding of the structural basis and mechanism of its function has become the focus of research.Ubiquitin proteolysis system is an important way to regulate protein degradation.Because of the abnormal degradation,the accumulation of cancer protein often promotes the occurrence and development of tumor.Therefore,ubiquitin ligase is considered as an important target for molecular targeted therapy.Up to now,a number of ubiquitin ligase inhibitors have been reported to have good antitumor effects,such as Mdm2(Mouse,double,minute,2)and NEDD8-Activating,Enzyme(NAE)inhibitors.RNF126(RING finger protein 126),an E3 ubiquitin ligase,is important in cell survival,development,cell growth and cancer biology by regulating different substrates.Up to now,the possible role of RNF126:in human glioma is still unclear.So we investigate the influence of RNF126 on the proliferation of glioma cells and the underlined mechanism.Part one:The study of the expression and correlation of RNF126 and p27 in gliomas and normal brain tissueObjective In this study,the expression of RNF126 and p27 in normal brain tissue and human glioma tissues are analyzed,and we also analyzed the relationship between expression and pathological grade of glioma.Methods This experiment is divided into normal brain tissue group,glioma grade ? group and ? class group.The normal brain tissue comes from the non necrosis normal brain tissue specimens of patients with traumatic brain injury who need intracranial decompression surgery.Western and Blot were used to detect the protein levels of RNF126 and p27 in 7 cases of human brain glioma specimens and 7 normal brain tissues,and statistical analysis was performed.The correlation between the two gene expressions was analyzed.In addition,the expression of RNF26 in glioma cells with different malignant degree was detected by Western Blot method.Result(1)We firstly studied the clinical relevance of RNF126 as well as the cell cycle negative regulation factor p27 and found that RNF126 was up-regulated in the human glioma tissues(P<0.01),which was reversely related to the level of p27(r=-0.71).In addition,we analyzed the expression of RNF126 in glioblastoma cell lines with different malignant degrees,and also showed that RNF26 was differentially expressed in glioma cells of different malignant extent.Conclusion(1)RNF126 and p27 are expressed in non tumorous brain tissues and brain glioma tissues.The expression of RNF126 in gliomas is significantly higher than that in non tumor tissues,and negatively correlated with the cell cycle negative regulator p27.(2)The expression of RNF26 is even higher in malignant glioma cell lines.Part two:Research on the mechanisms of RNF126 in glioma cell proliferationObjective To investigate the effect of RNF126 on the proliferation of glioma cells and its possible mechanism,and to provide potential molecular targets for the molecular targeted therapy of glioma.Methods(1)Molecular cloning technique was used to construct LV-shRNF126 and LV-GFP-RNF126 plasmids,and packaging lentivirus to infect glioma cells,and to construct stable silent and overexpressed RNF126 cell lines.(2)In the stable cell line constructed,EdU infiltration assay,colony forming assay and MTT assay were used to detect the proliferation of silent and over expressed RNF126 glioma cells.(3)The interaction of RNF126 and p27 was detected by irnunoprecipitation and Western blot,and the protein level and ubiquitination level of p27 were detected.Results(1)The silencing efficiency of RNF126 was detected by Western blot,and the effect of RNF126 silencing in transfected pLV-shRNF126#1 group was more than 80%by Western blot analysis.The application of LV-shRNF126 and LV-GFP-RNF126 plasmids lentiviral infection of U251 glioma cells,immunofluorescence observation showed that the infection efficiency was above 90%and the silencing effect is good,suggesting that U251-shRNF126 silencing cell line was successfully constructed.(2)In order to verify the importance of RNF126 on the proliferation of glioma cells,we conducted experiments with EdU U251-shRNF126 and U251-control cell lines,EdU experimental results show that a low proliferation rate of U2 51-control 57%U251-shRNF126 proliferation rate.At the same time,U251-shRNF126 and U251-control cell lines were used for colony forming assay.The statistical result showed that the ability of U251-shRNF126 cell formation was lower than that of U251-control.In addition,we carried out MTT experiments with U251-shRNF126 and U251-control cell lines,and the results of MTT experiments were consistent with the results of EdU and colony forming experiments.The results showed that the proliferation of cells after silencing RNF126 was significantly reduced.These results indicate that the proliferation of RNF126 cells is decreased after silencing RNF126,so what is the change in the proliferation ability of glioma cells after overexpression?In order to further investigate the effect of RNF 126 on cell proliferation,we packed GFP-RNF126 and GFP lentivirus and constructed U251-GFP-RNF126 and U251-GFP cell lines by lentivirus infecting U251 cells.Immunofluorescence experiments showed that the infection rate was above 90%.In order to detect the expression of exogenous RNF 126 in U251-GFP-RNF126 cells,we extracted U251-GFP-RNF126 and U251-GFP protein by Western Blot assay showed that RNF126 can be stably expressed in U251 cells,indicating that U251-GFP-RNF126 and U251-GFP cell lines was successfully constructed.To further validate the role of RNF 126 in promoting glioma cell proliferation,we performed EdU experiments on U251-GFP-RNF126 and U251-GFP cell lines.EdU detection showed that the proliferation rate of U251-GFP-RNF126 cells was 110%higher than that of U251-GFP cells.The cell colony forming ability was detected by U251-GFP-RNF126 and U251-GFP cell lines.The statistical result showed that the ability of U251-GFP-RNF126 cell clone formation was significantly higher than that of U251-GFP cell line.The MTT experiment also showed that the proliferation ability of U251-GFP-RNF126 and U251-GFP was stronger than that of the control group.These results suggest that RNF 126 may play an important role in the proliferation of human brain glioma cells.Silencing RNF126 significantly inhibits the proliferation of glioma cells.Overexpression of RNF126 can significantly promote the proliferation of glioma cells.(3)To further explore whether RNF126 can promote the proliferation of glioma cells molecular mechanism by which,we first in U251 cells after FLAG-RNF126 transfection technology to verify the interaction between p27 and RNF126 by CO immunoprecipitation.The results showed that overexpression of RNF126 in U251 cells could be combined Avith p27.Further,endogenous protein Co-IP proved that endogenous RNF26 also interacts with p27.(4)We then found that silent RNF126 significantly up-regulated the protein level of p27,whereas overexpression of RNF26 significantly lowered the protein level of p27.Accordingly,silencing RNF126 significantly attenuated ubiquitination of p27,whereas overexpression of RNF26 significantly increased ubiquitination of p27.Finally,we demonstrate that RNF126 mediated p27 degradation can be blocked by proteasome inhibitor MG 132.We also attempted to do an in vitro ubiquitination assay,but we did not detect an in vitro ubiquitination of p27.This suggests that there may be other factors involved in RNF126 mediated ubiquitination of p27,such as modifications of p27,or some other adaptor proteins.In conclusion,our study demonstrates the high expression of RNF26 in glioma proliferation and promotes the proliferation of glioma cells by negatively regulating the cell cycle inhibitor p27.Conclusions(1)Down-regulation of RNF126 suppressed the proliferation of human glioma cells,while overexpression of RNF126 promoted the proliferation of human glioma cells.(2)RNF126 promoted glioma cell proliferation by regulating the ubiquitination and degradation of D27.