Study On The Mechanism Of Mucin O-glycosylating Enzyme GALNT2 Influencing The Malignant Characteristics Of Glioma

BackgroundGliomas account for 80%of all primary brain tumors.More than 50%of them were glioblastomas(GBM).Current multimodality therapy includes maximal surgical resection,radiotherapy,chemotherapy and immunotherapy.However,the therapeutic result,which is measured by overall survival and survival quality,remains unsatisfactory.With the development of molecular biology and tumor genetics,the understanding of glioma pathogenesis is deepening.Genomic alterations,such as mutations in the isocitrate dehydrogenase(IDH)gene,codeletion of lp/19q,methylation of the O6 methylguanine methyltransferase(MGMT)gene promoter,and mutations/amplifications of the epidermal growth factor receptor(EGFR)have been confirmed to be closely related to glioma tumorigenesis.Therefore,the molecular alterations that occur in glioma are expected to help predict prognosis and provide better therapeutic strategies.Glycosylation is one of the most common event in the posttranslational modification of proteins.The modified protein accounts for more than 70%of the total protein in cells.There are two major types of protein glycosylation:N-linked and O-linked.The mucin type is the most common type of O-glycosylation.GALNT2 is the enzyme that regulates the initial step of mucin O-glycosylation.Aberrant glycosylation can influence multiple cellular properties,including cell proliferation,transformation,differentiation,apoptosis,migration and invasion.Previous studies have revealed that GALNT2 correlates with the activation of receptor tyrosine kinases(RTKs),which are important biological receptors with ligand-binding and protein kinase activity.Among them,EGFR was the first to be reported and the most well-studied RTK for its roles in signal transduction and cancer progression.Previous studies have revealed that EGFR may express short O-glycans.Aberrant GALNT2 expression has been reported to influence the malignancy of various cancers.For instance,GALNT2 can modify EGFR glycosylation and activity and thereby regulate the malignant behavior of HCC cells.The downregulation of GALNT2 modifies the malignant progression of gastric cancers by increasing MET phosphorylation and influencing the EGFR glycosylation and activation.However,the function of GALNT2 has not previously been reported in glioma cells.Based on the current researches,we combined with the results of differential expression analysis in TCGA database to illustrate the expression characteristics and prognostic clinical significance of GALNT2 in glioma.Firstly,GSEA was used to predict the biological functions and possible signal pathways significantly related to GALNT2.Then the specific mechanism of its action was elaborated through functional experiments and Western blot verification,and in vivo verification was carried out through animal experiments,in order to provide new ideas and targets for clinical treatment of glioma.Objectives1.To explore the expression level of GALNT2 in different grades of glioma and its clinical prognostic significance.2.To investigate the malignant phenotype of GALNT2 in the biological behavior of glioma through the functional experiments of glioma cells.3.To explore the specific molecular biological mechanism of GALNT2 through which affecting the malignant phenotype of glioma.4.To further verify the effect of GALNT2 on malignant progression of glioma in vivo through the animal experiments.Methods1.Bioinformatics techniques were used to explore the expression characteristics of GALNT2 in glioma and to plot the survival curve,and to predict its related biological functions and possible signaling pathways.①The expression characteristics and survival curves of GALNT2 in glioma were plotted by TCGA database of public bioinformatics platform.②GSEA was used to predict the related biological functions and possible signal pathways of GALNT2.2.GALNT2 expression level of normal brain tissue and the WHO(Ⅱ-Ⅳ)glioma specimens was examined.The protein levels of GALNT2 were examined via immunohistochemistry in human gliomas(n=35)and normal brain tissues(n=5)from Qilu Hospital(Jinan,China).Paraffin-embedded glioma tissues(WHO Ⅱ-Ⅳ)were obtained from patients(n=35)who underwent surgery at the Department of Neurosurgery,Qilu Hospital of Shandong University.Normal brain tissue samples(n=5)were collected from patients with severe traumatic brain injury who underwent partial resection of the normal brain.3.The effect of GALNT2 knockdown on the proliferation,cell cycle,invasion and migration of glioma cells was studied.① RNA interference technique was used to instantaneously transfect human glioma U251 and U87MG cell lines,and the knockdown efficiency was detected by real-time quantitative PCR and Western blot.② The effect of GLANT2 knockdown on proliferation and activity of U251 and U87MG cells was detected by EdU and CCK-8.③ The effect of GLANT2 knockdown on glioma cell cycle was detected by flow cytometry.④The effects of GLANT2 knockdown on the invasion and migration of glioma cells were tested by 3D spherification experiment,Transwell migration experiment,Matrigel invasion experiment and Wound healing experiment.4.The effect of GALNT2 overexpression on proliferation,cell cycle and invasion and migration of glioma cells was explored.①Plasmid overexpression technique was used to instantaneously transfect human glioma U251 and U87MG cell lines,and the overexpression efficiency was detected by Western blot.②EdU,cck-8,flow cytometry,Transwell migration assay and Matrigel invasion assay were used to examined the effects of GALNT2 overexpression on glioma cell proliferation,cell cycle,invasion and migration.5.The molecular biological mechanism of GLANT2 regulating the proliferation,invasion and migration of glioma cells was studied.① According to GSEA prediction of GALNT2-related signaling pathways,Western blot and immunoprecipitation were used to verify the molecular mechanism of GALNT2 affecting the proliferation and invasion and migration ability of glioma cells.②The mechanism of its regulation in the signal pathway was further confirmed by using enhancers and inhibitors of relevant key molecules in the signal pathway respectively.6.The effect of GALNT2 on the proliferation and invasion and migration of glioma cells in vivo was verified by animal experiments① Lentivirus vector packaged with sh-GALNT2 was transfected into U87MG fluorescence cells and intracranial orthotopic model of human glioma tumor bearing nude mice was established.② The size of intracranial tumors was measured regularly by bioluminescence imaging,and the survival and weight of the mice were recorded.③ HE staining was used to check the tumor boundary and immunohistochemistry was used to detect its effect on related molecules.Results1.High GALNT2 expression is associated with worse patient survival.Patients with high GALNT2 expression(>median value)exhibited a significantly worse prognosis than those with low GALNT2 expression among all glioma patients(P<0.0001).2.Increased GALNT2 expression is associated with increased tumor grade and worse subtypes in glioma.GALNT2 mRNA levels were significantly increased in GBMs compared with those in normal brain tissues and LGGs in TCGA.GALNT2 expression was upregulated in LGGs,according to WHO grade.GALNT2 expression was substantially higher in the mesenchymal subtype than in the proneural subtype in the TCGA database.High GALNT2 expression was significantly associated with clinicopathological characteristics of patients,such as patient age(≥45;P=0.001).Molecular genetic features,including IDH mutation,MGMT promotor methylation and codeletion of lp/19q,have been shown to be associated with better prognosis in gliomas.Low GALNT2 expression was associated with these features(P<0.001).3.Potential biological functions and pathway analysis of GALNT2.The results of GSEA based on the GALNT2 expression in the TCGA database showed that GALNT2 was concentrated in cancer-related pathways.Moreover,high GALNT2 expression was associated with the epithelial-mesenchymal transition(EMT),extracellular matrix(ECM)receptor interactions,O-glycan biosynthesis and the EGFR.In pathway analysis,we found that the PI3K/Akt/mTOR signaling pathway positively correlated with high GALNT2 expression in gliomas.4.Knockdown of GALNT2 induces cell cycle arrest in human glioma cells.① qRT-PCR and Western blot were used to verify the knockdown efficiency.EdU and CCK-8 assays were performed.GALNT2 downregulation resulted in a significant decrease in the percentage of EdU-positive cells,as well as the OD450 values,in both U87MG and U251 cells at 48 h after transfection.Cell cycle analysis showed that GALNT2 downregulation increased the population of glioma cells in the G0/G1 phase.Furthermore,the colony-formation assay was used to evaluate the long-term effects of sh-GALNT2 on cell proliferation.The results showed that sh-GALNT2-transfected cells formed significantly fewer colonies than the control group.②GALNT2 knockdown significantly decreased the level of phosphorylated Akt and mTOR,which are essential for cancer progression in various cancers including gliomas,while the expression of total Akt and mTOR exhibited no obvious change.In addition,cyclin-dependent kinase 4(CDK4)and cyclinD1 expression were decreased after GALNT2 silencing.In contrast,the cyclin-dependent kinase inhibitor p21,a tumor suppressor,was increased in the knockdown group.In summary,these results indicated that GALNT2 downregulation suppressed cell cycle progression in glioma cells.5.Knockdown of GALNT2 inhibits glioma cell migration and invasion①3D collagen spheroid invasion assay showed silencing GALNT2 reduced the area invaded by U87MG and U251 spheroids relative to controls.In the transwell migration assay,the number of migrated glioma cells in the GALNT2-knockdown group was significantly decreased compared with that in the control group.Transwell invasion assays revealed that compared with the control groups,GALNT2 downregulation significantly reduced the number of glioma cells that invaded through the Matrigel-coated membrane at 48 h.The wound-healing assays demonstrated that the migratory ability of glioma cells was significantly suppressed following transfection with si-GALNT2.②GALNT2 knockdown induced the downregulation of MMP2 and MMP9 proteins in U87MG and U251 cells.These results suggested that GALNT2 knockdown significantly inhibits glioma cell migration and invasion in vitro.6.GALNT2 overexpression promotes glioma cell proliferation,migration and invasionWestern blot analysis verified that the protein levels and the expression of p-Akt,MMP2,CDK4,and cyclin D1 were increased,while those of p21 were decreased,in the GALNT-overexpression group.GALNT2 upregulation resulted in an increase in the percentage of EdU-positive cells and OD450 values in both U87MG and U251 cells.Cell cycle analysis also demonstrated that GALNT2 overexpression decreased the population of the U87MG and U251 cells in the G0/G1 phase.In transwell migration assays,the number of migrated glioma cells in the GALNT2-overexpression group was increased compared with that in the control group.The invasion assays revealed that compared with the control groups,GALNT2 upregulation significantly increased the number of glioma cells that invaded through the Matrigel-coated membrane at 48 h.These results showed that the overexpression group of GALNT2 had an opposite effect compared with the knockout group,and could significantly promote the proliferation,migration and invasion ability of glioma cells.7.Knockdown of GALNT2 decreases EGFR phosphorylation and modified EGFR O-glycosylationGALNT2 knockdown decreased the EGF-induced phosphorylation of EGFR in U87MG and U251 cells.The VVA agarose beads could detect the expression of Tn antigen(GalNAc-O-Ser/Thr)on EGFR.The result showed that GALNT2 knockdown reduced VVA binding to EGFR,which implied that GALNT2 knockdown decreased the O-glycosylation of EGFR.8.GALNT2 facilitates glioma progression through the PI3K/Akt/mTOR signaling pathway① The knockdown group was treated with an Akt activator(SC79).The expression of p-Akt,p-mTOR,and cell cycle regulatory factors were increased,while that of p21 decreased,compared with the group without the activator.CCK-8 assays showed that after treatment with the Akt activator,the knockdown group exhibited an increase in proliferation ability compared with the group treated with DMSO.The wound-healing assays demonstrated that the migratory ability of GALNT2-knockdown cells was also increased following treatment with the Akt activator compared with DMSO.② The overexpression group was treated with a PI3K inhibitor(LY294002).The expression of p-Akt,p-mTOR,and cell cycle regulatory factors was decreased,while that of p21 increased,compared with the group without the inhibitor.The results of CCK-8 and wound-healing assays showed that the proliferation and migration ability of the GALNT2-overexpression group was inhibited after treatment with the PI3K inhibitor.These data indicated that GALNT2 delivered signals through the PI3K/Akt/mTOR signaling pathway.9.GALNT2 downregulation inhibits the tumorigenesis and aggressiveness of glioma cells in vivo① Animals bearing sh-GALNT2 cells displayed significantly reduced tumor size via bioluminescence imaging.The sh-GALNT2 group exhibited a longer survival rate relative to controls,and its weight loss was substantially slower than that in the control group.② HE staining of tumors that were collected at the same time(14 days after implantation)in the two groups also showed a smaller size in the sh-GALNT2 group,and tumors of the sh-GALNT2 cells displayed significantly more circumscribed bordersc.IHC confirmed that GALNT2 protein levels were reduced in xenografts generated with sh-GALNT2 cells.The proliferation index marker Ki-67 and the invasion marker MMP2 were also decreased in sh-GALNT2 xenografts.These results demonstrated that GALNT2 knockdown led to reduced growth and invasion of glioma cells in vivo.ConclusionIn conclusion,our study highlighted that increased GALNT2 expression levels were associated with an unfavorable prognosis and a higher tumor grade in human gliomas.GALNT2 facilitated glioma cell proliferation,migration and invasion by influencing EGFR O-glycosylation and phosphorylation and the downstream PI3K/Akt/mTOR pathway in vitro and in vivo.Therefore,GALNT2 may serve as a novel biomarker and a potential therapeutic target for human glioma.