High Expression Of TAGLN2 And RAB43 Predict Poor Prognosis And Contribute To EMT Process In Gliomas

BackgroundGliomas have been reported to be the most common type of primary intracranial tumors and account for approximately 88%of all malignant brain tumors.According to the World Health Organization(WHO),gliomas are classified into four malignancy grades(I-IV)based on their histological features,among which glioblastoma multiform(GBM,grade IV)shows the worst prognosis,with a median survival time of only 12-15 months and a 5-year survival rate<5%in glioblastoma patients.Generally,completely surgical resection of tumor is the main treatment of malignant gliomas.However,some of the patients still suffer from tumor recurrence,resulting in an unsatisfactory survival rate,which suggests that there still remain molecular factors promoting the invasion and metastasis of tumor cells to infiltrate the normal brain parenchyma.Therefore,it is essential to investigate the underlying molecular mechanism and search for novel biomarker that will facilitate the diagnosis and treatment of gliomas.With advancement of molecular biology technology,new methods for classifying gliomas have emerged in recent years.For the past century,the classification of brain tumors has been based largely on concepts of histogenesis that tumors can be classified according to their microscopic similarities with different putative cells of origin and their presumed levels of differentiation.The characterization of such histological similarities has been primarily dependent on light microscopic features in hematoxylin and eosin-stained sections,immunohistochemical expression of lineageassociated proteins and ultrastructural characterization.The 2016 CNS WHO presents major restructuring of the diffuse gliomas,medulloblastomas and other embryonal tumors,and incorporates new entities that are defined by both histology and molecular features,including IDH1 mutation,methylation of O-methylguanine-DNA methyltransferase(MGMT)promoter,co-deletion of 1p/19q,telomerase reverse transcriptase(TERT)loss,and alpha thalassemia/mental retardation syndrome X-linked(ATRX)mutation.Based on mRNA expression microarray,The Cancer Genome Atlas(TCGA)described a robust gene expression-based molecular classification of GBMs that divided them into four subtypes:proneural,neural,classical,and mesenchymal,among which the mesenchymal subtype was distinguished from the others as being particularly malignant.Thus,there is an urgent need for the exploration of novel biomarkers and therapeutic targets for GBM molecularly classified as the mesenchymal subtype.Glioma progression is a dynamic process in which EMT is a key event driving invasion of tumor cells..Several EMT-related factors have been previously associated with increased invasion and poor prognosis in gliomas.It has been reported that EMT occurs during tumor progression,leading to increased motility and invasiveness of cancer cells.An obvious characteristic of EMT is up-regulation of the mesenchymal markers such as N-cadherin,Vimentin,and Snail.Transgelin-2(TAGLN2)is an actin-cross-linking protein containing a calponin homolog(CH)domain,with a molecular weight of 24 kDa.It has been reported that TAGLN2 is involved in the regulation of cell transformation and cell morphology.More recently,the dysregulation of TAGLN2 in a variety of malignant tumor types,including colorectal cancer,bladder cancer,lung cancer,uterine cervical squamous cell carcinoma,and breast cancer,has been discovered through proteomic analysis,and thus reveals an important role for TAGLN2 in tumor progression.The expression pattern and clinical significance of TAGLN2 in human gliomas,however,have not been determined.Furthermore,it remains unknown as to whether TAGLN2,because of its role in cell transformation and cell morphology,is involved in the regulation of the epithelial-mesenchymal transition(EMT).Ras-related GTP-binding protein 43(RAB43)is a member of the Ras superfamily with a molecular weight of 23 kDa.Previous studies have shown that RAB43 is mainly located in endoplasmic reticulum and Golgi,and plays a role as a key regulator of vesicle movement,signal transduction and tethering membrane events in membrane trafficking.Recent studies revealed that some of the RAB proteins are involved in the regulation of several signal transduction pathways relating to cell invasion,cell apoptosis and innate immune response.Meanwhile,several members of RABs including RAB IB,RAB3D,RAB27A and RAB38 have been demonstrated to be dysregulated in a variety of malignant diseases and playing critical roles in tumor progression.Nonetheless,differential expression,intracellular function and potential mechanism of RAB43 in tumors have not been reported.Here,we used publicly available datasets to determine the pattern of TAGLN2 and RAB43 expression in human gliomas,and its relationship with tumor grade,and other clinicopathological indicators and molecular features of gliomas.Their function were investigated both in vitro and in vivo as well as potential pathways regulating it.Our findings indicate that TAGLN2 and RAB43 might be significant prognostic indicator and a potential therapeutic target for human gliomas.Part ?TAGLN2 is a candidate prognostic biomarker promoting tumorigenesis in human gliomasObjectivesTransgelin-2(TAGLN2)is a member of the calponin family of actin-bundling proteins that is involved in the regulation of cell morphology,motility,and cell transformation.Here,the clinical significance and potential function of TAGLN2 in malignant gliomas were investigated.MethodsMolecular and clinical data was obtained from The Cancer Genome Atlas(TCGA)database.Gene ontology and pathway analysis was used to predict potential functions of TAGLN2.RNA knockdown was performed using siRNA or lentiviral contructs in U87MG and U251 glioma cell lines.Cells were characterized in vitro or implanted in vivo to generate orthotopic xenografts in order to assess molecular status,cell proliferation/survival,and invasion by Western blotting,flow cytometry,and 3D tumor spheroid invasion assay,respectively.Results1.Increased expression of TAGLN2 is associated with increasing tumor grade in gliomaTAGLN2 mRNA levels were significantly increased in GBMs compared to LGGs and normal brain tissues in TCGA(P<0.001).The expression of TAGLN2 was also up-regulated in LGGs relative to normal brain(P<0.001).TAGLN2 was high in the mesenchymal subtype in TCGA,CGGA and GSE4271 databases and low in the proneural subtype.Patients with wild type IDH1 exhibited higher expression of TAGLN2 than those with mutated IDH1.Low TAGLN2 was also associated with other molecular characteristics,including methylated MGMT,lp/19q codeletion,loss of TERT and mutated ATRX in tumors(P<0.001;respectively).2.TAGLN2 expression is associated with poorer patient survivalHigh TAGLN2 expression(>median value)had a significantly worse prognosis than those with low TAGLN2 expression in LGG(78.2 vs 114.0 months,P<0.001)and GBM patients(12.5 vs 22.2 months,P<0.05).Furthermore,TAGLN2 expression was validated as an independent indicator of OS after multivariate Cox regression analysis(HR = 1.713,95%CI = 1.384 to 2.119,P<0.0001).TAGLN2 might therefore be a novel prognostic biomarker in gliomas.3.Pathway analysis of TAGLN2 and co-regulated genesGene ontology(GO)analysis indicated that TAGLN2 was tightly associated with many biological processes,including signal transduction,cell survival,cell adhesion,and extracellular matrix organization,as well as cell cycle transition.KEGG analysis demonstrated that TAGLN2 was enriched in pathways in cancer,focal adhesion and regulation of the actin cytoskeleton.Finally,GSEA analysis revealed that high levels of TAGLN2 were significantly related to EMT,cancer metastasis,and the G1-S phase transition of cell cycle progression.4.TAGLN2 silencing reduces invasion and inhibits mesenchymal properties in glioma cells in vitro3D collagen spheroid invasion assay showed that silencing TAGLN2 with a single siRNA reduced the area invaded by U87MG and U251 spheroids.Western blot analysis revealed that knockdown of TAGLN2 led to decreases in several mesenchymal factors,including N-cadherin,?-catenin,Snail,Slug,and Twist,and significant increases in the epithelial marker(E-cadherin)compared to controls.5.Knockdown of TAGLN2 induces cell cycle arrest and apoptosis in human glioma cellsDown-regulation of TAGLN2 resulted in a statistically significant decrease in OD450 values as well as the percentage of EdU positive cells in both U87MG and U251.TAGLN2 knockdown significantly decreased the level of Fork head box M1(FoxMl).In addition,cyclin-dependent kinase 2(CDK2),cyclin B1,and cyclin D1 expression were decreased after TAGLN2 silencing.Furthermore,a marked reduction in downstream targets of p21,c-Myc and Survivin,was observed after TAGLN2 knockdown.Finally,TAGLN2 silencing promoted apoptotic cell death by?three-and two-fold compared with controls in U87MG and U251 cell lines6.TGF?2 induces TAGLN2 in glioma cell linesTGF?2 was higher in GBM than in LGG,just as for TAGLN2,and patients with high expression of both TGFP2 and TAGLN2 exhibited significantly poorer OS.Addition of TGF?2(5 ng/mL)to cell culture led to increases in TAGLN2 protein levels by?two-and three-fold in U87MG and U251 cell lines.In contrast,TAGLN2 was decreased upon exposure of cells to the TGF?/Smad pathway specific inhibitor SB431542(5?M).7.TAGLN2 silencing inhibits tumor growth in vivoOrthotopic xenografts were established by implanting U87MG-sh-TAGLN2 or U87MG-control cells into nude mice.Animals bearing U87MG-sh-TAGLN2 cells displayed significantly reduced tumor size and increased survival relative to controls.The EMT classical marker N-cadherin and the proliferation index marker Ki-67 were also decreased in U87MG-sh-TAGLN2 xenografts.ConclusionsOur findings indicate that TAGLN2 has a role in promoting the development of human glioma.The regulation and function of TAGLN2 therefore renders it as a candidate molecular target for the treatment of GBM.Part ? High expression of RAB43 predicts poor prognosis and associates with epithelial-mesenchymal transition in gliomasObjectivesRas-related GTP-binding protein(RAB)family play an important role in regulating signal transduction and cellular processes including vesicle transport,cytoskeleton formation,and membrane trafficking.More recently,several RAB members have been reported to promote tumorigenesis in many types of cancers.However,the clinical significance and potential function of RAB43 in gliomas remain unclear.MethodsExpression of RAB43 was analyzed by in silico analysis and immunohistochemistry(IHC).Gene ontology and pathway analysis was used to predict potential functions of RAB43.RNA knockdown was performed using siRNA in U87MG and U251 glioma cell lines.Cells were characterized in vitro in order to assess molecular status,cell proliferation/survival,and invasion by Western blotting,flow cytometry,and Transwell invasion assay.Results1.Expression of RAB43 was associated with glioma grade progressionRAB43 expression tends to increase from grade II to grade IV according to the WHO classification(p<0.0001).Furthermore,high expression of RAB43 was statistically associated with patients with age?45(p=0.0056).It has been demonstrated that glioma patients with Karnofsky Performance Status(KPS)score>80 had a better prognosis than those with a KPS of<80.However,no significant differences were identified between RAB43 expression in relation to KPS score(p=0.0675)2.High RAB43 expression was related to poor clinical outcomes in gliomasWe found that patients with high RAB43 expression had a significant shorter overall survival time than those with low RAB43 expression.Moreover,the prognostic values of RAB43 in GSE16011(n=276)and Rembrandt databases(n=329)were analyzed,and the results indicated that high RAB43 was significantly associated with worse OS both in low-and high-grade glioma patients.3.RAB43 expression showed a subtype preferenceHigh RAB43 level was associated with mesenchymal subtype and G3 subtype.In contrast,patients with low RAB43 were more likely to be proneural subtype.Patients with IDH1 gene wild-type showed higher expression of RAB43 than those with mutant IDH1.Additionally,RAB43 was observed to be up-regulated in patients with unmethylated MGMT,lp/19q non-co-deletion,but not with ATRX gene wild-type gliomas.4.RAB43 was significantly associated with tumor cell adhesion and invasionGO analysis revealed that RAB43 was strongly associated with biological processes including response to stimulus,cell adhesion,cell migration,extracellular matrix organization as well as response to wounding.In KEGG analysis,the up-regulated genes were enriched in pathways related to focal adhesion,cell adhesion molecules(CAMs)and ECM-receptor interaction in cancer progression.There was a significant positive correlation between RAB43 and MMP-2,MMP-9,interleukin-6(IL-6),IL-8,vascular endothelial growth factor A(VEGFA),Ki-67 and several mesenchymal subtype markers(Vimentin,N-cadherin and Snail).5.RAB43 silencing reduced glioma cell migration and invasionSilencing RAB43 significantly inhibited both migration and invasiveness of U87MG glioma cells,down-regulation of MMP-2 and MMP-9 level were observed.Moreover,RAB43 knocking down significantly suppressed expression of the mesenchymal markers(N-cadherin,Vimentin),as well as EMT-related transcription factor Snail in both U87MG and U251 cells.ConclusionsHigh level of RAB43 is significantly associated with the malignant phenotypes of gliomas,which suggests that RAB43 may serve as a novel biomarker and a potential therapeutic target for gliomas.