Effects And Mechanisms Study Of MiR224-3p And MIF In Glioblastoma Under Hypoxia Microenvironment

BackgroundGlioblastoma Mutiforme(GBM)is the most common and aggressive malignant primary tumor of the adult central nervous system,accounting for more than 50%of all gliomas.According to histopathologic features,GBM is defined as the highest grade(grade ?)astrocytoma by the World Health Organization(WHO)classification of tumors of the central nervous system.In recent years,although progresses in therapeutic strategies,for instance,combining surgery with chemo-radiotherapy,anti-angiogenic therapy and immunotherapy,the recurrence rate of GBM is very high and the prognosis of patients with GBM is poor,around 12 months of the median survival time.In order to complete and supplement the current therapeutic strategies,it is urgent to further explore and reveal the molecular mechanisms of the malignant progress of glioblastoma.Hypoxia is a hallmark feature of the solid tumor microenvironment.It has been estimated that 50 to 60%of solid tumors contain regions of hypoxia and/or anoxia that arise as a result of an imbalance between oxygen delivery and oxygen consumption.Within the tumor microenvironment,oxygen delivery is impaired because of abnormalities in the tumor vasculature characterized by leaky and sluggish blood flow.At the same time,oxygen consumption rates are high because of the demands of proliferating tumor cells and infiltrating immune cells.Hypoxia and HIF-dependent signaling play an important role in tumor malignant progression.Hypoxia participates in regulation of epithelial-mesenchymal transition(EMT),invasion,migration,angiogenesis,vasculogenic mimicry and immune escape of tumor cells.In addition,clinical data suggest that hypoxia is associated with metastasis,resistance to chemotherapy and radiotherapy,as well as poor patient survival.As a malignant solid tumor,GBM contains a wide range of hypoxic region.However,the molecular regulation mechanisms of GBM under hypoxia has not yet fully elucidated.A better understanding of the molecular mechanism could help us to find new therapeutic targets and bring novel treatment strategies to prohibit the malignant progress of GBM to improve the prognosis of glioma patients.Autophagy is a self-digesting and catabolic process that recovers damaged,denatured or senescent proteins and organelles in cells.The process of metabolic waste self-elimination and energy recovery play an important role in the normal operation,self-repair and resistance to harsh environments of cells.More importantly,autophagy induced by harsh environments such as hypoxia,ischemia,starvation,etc.provides an effective way for tumor cells to enhance the viability and promote the malignant progress of the tumor.Studies have shown that hypoxia can induce autophagy of GBM cells and improve its resistance ability to drug therapy.MiRNA(MicroRNA)is a class of 19-22nt non-coding,single-stranded RNA that bind on the 3 ' untranslated region(3'UTR)of target genes to cause degradation or inhibition of translation of the target genes' mRNA,regulating the genes expression at post-transcriptional level.MiRNAs play an important role in various pathophysiology,for exa:mple:cel]proliferation,differentiation,apoptosis and autophagy.In GBM,hypoxia microenvironment induced a large number of miRNAs to produce differential expression,leaving a specif-ic hypoxia signature of miRNA expression profile.These differentially expressed miRNAs may play a critical role in hypoxia-induced autophagy,invasion and migration and the generation of drug resistance ability in GBM cells.However,the key targets of hypoxic miRNAs on autophagy and malignant progression of GBM are still unclear and require further study.Vasculogenic mimicry(VM)is de novo vascular networks formed by malignant cancer cells themselves.Since the initial conceptualization of tumor cell VM,it was identified in various malignant tumors,including GBM.VM could provide increasing amounts of blood flow for nutrient and oxygen supply and is essential for the malignant behavior of gliomas.Recent study showed that tumor cells that are capable of VM share a plastic phenotype that could be regulated by hypoxia.Consistently,hypoxia-induced EMT of GBM is strongly associated with the formation of VM.These findings suggested that there is close relation between hypoxia and VM formation.Macrophage migration inhibitory factor(MIF)is a cytokine with pro-inflammatory and tumor-promoting functions.MIF is highly expressed in various types of tumors,including GBM.In recent years,the significant role of MIF in tumorigenesis of GBM has been revealed.MIF expression is correlated with the malignant progress and infiltration of GBM.More importantly,the expression and secretion of MIF is strongly induced by hypoxia and MIF is closely associated with autophagy,migration and invasion,angiogenesis and immune escape in GBM.However,both the specific role and regulatory mechanisms of MIF on GBM cells in hypoxia environment remain to be explored.In this study,we investigated the main roles and molecular mechanisms of miR224-3p and MIF in autophagy and vasculogenic mimicry of GBM under hypoxia.First,the miRNA expression profile of GBM in hypoxia was analyzed.MiR-224-3p,which was strongly down-regulated by hypoxia,was found to be involved in the regulation of autophagy in GBM.Furthermore,we revealed its detail role and molecular mechanism in GBM.In addition,we found that MIF played an important role in regulation of hypoxia-induced vasculogenic mimicry of GBM,and further explored its specific molecular mechanism.Our basic research of GBM under hypoxia provided new potential therapeutic targets for treatment of GBM and has important theoretical value and clinical significance.Part I:The effect and mechanism of miR224-3p on hypoxia-induced autophagy in GBM cells1.Objective(1)Based on miRNA microarray,through analysis of miRNA expression profiles of GBM cell line under normoxia and hypoxia,to screen hypoxia down-regulated miRNAs and find out autophagy related miRNAs(miR224-3p).(2)To clarify the role of miR224-3p on the autophagy activity of GBM cells under hypoxia;to elucidate the mechanism of miR224-3p regulating autophagy in GBM cells and the direct targeting genes of miR224-3p.(3)To reveal the expression level of miR224-3p in glioma tissues and the correlation between miR224-3p and target gene expression.(4)To investigate the effect of miR224-3p on cell proliferation and hypoxia-induced apoptosis in GBM cells.(5)To verify the effect of miR224-3p on tumorigenic growth of GBM cells.2.Method(1)Hypoxia treatment and miRNA microarray Hypoxia treatment was performed by incubating cells in an incubator chamber flushed with a gas mixture containing 1%02,5%CO2 at 37?.The total miRNAs were extracted from U251 cells exposed to hypoxia or normoxia for 24 h.Then the comprehensive miRNA expression profiles were detected and analyzed.(2)Detection of mRNA,miRNA and protein levelsClinical glioma tissue specimens of different pathological grades were collected to extract total mRNAs and prepare paraffin sections.Then the expression levels of miRNAs and proteins were measured by quantitative real-time PCR(q-PCR)and immunohistochemistry.The U251 and U87 cell lines treated with special conditions were extract to gain total mRNA and protein.Then the expression levels of mRNAs,miRNAs and proteins in U251 and U87 cells were detected by q-PCR and Western blot.(3)Cell transfection and construction of GFP-LC3B stable cell lines U251 and U87 cells were transiently transfected with miR224-3p mimics,miR224-3p inhibitor,ATG5-overexpression plasmid,FIP200-overexpression plasmid,ATG53'UTR binding domain mutant or wild luciferase reporter plasmid and FIP200 3'UTR binding domain mutant or wild luciferase reporter plasmids.Respectively transient transfection was used to overexpress or knock-down the corresponding proteins and miR224-3p,and then to study the changes of expression levels of various related indexes.The GFP-LC3B plasmid was packaged with lentivirus.U251 and U87 cell lines were stably infected with the lentivirus.Then the GFP-LC3B green fluorescent stable cell lines were constructed and used to monitor the autophagy activity.(4)Detection of autophagy activity in glioma cellsThe methods of detecting autophagy level are as below:? Transmission Electron Microscope(TEM)was used to directly observe the number of autophagosomes in U251 cells.This method is the gold standard of monitoring autophagy.? Western blot was used to detect the expressions of autophagy-associated proteins(LC3B and P62)in U251 and U87 cells.? Fluorescence microscope was used to detect the quantity and intensity of fluorescent autophagosome in U251 and U87 cells stably expressing GFP-LC3B.? Bafilomycin A1(BAF)was applied to assess autophagic flow.(5)Bioinformatics prediction of miRNA's target genes and luciferase reporter assayThe miRNA target gene databases(targetscan,PicTar,miRanda,etc.)were used to screen the autophagy-related target genes that miR224-3p was most likely to directly regulate.And then candidates were verified by q-PCR and Western blot.Finally,U251 and U87 cells were co-transfected with miR224-3p mimics and luciferase reporter plasmids containing the mutant or wild-type 3'UTR binding sites of the target genes,respectively,and double luciferase reporter assay was performed to determine miR224-3p direct target genes.(6)Glioma cell proliferation and apoptosis assayU251 and U87 cells were transiently transfected with miR224-3p mimics and miR224-3p inhibitor,respectively.The viability and proliferation of glioma cells were measured by CCK-8 and EdU kits.Hypoxia-induced apoptosis proportions of U251 and U87 cells were examined by AnnexinV-FITC Apoptosis Detection Kit and anti-cleaved-caspase3 immunocytochemistry.(7)Subcutaneous tumorigenesis of human GBM cell in nude miceU87 cells were inoculated subcutaneously into the flanks of nude mice to form tumors.Lentivirus-miR224-3p mimics were directly injected into each tumor.At the same time,tumor volume and terminal tumor weight of miR224-3p mimics group and control group were measured.3.Result(1)Hypoxia induces GBM cell autophagic activityThe autophagy level of U251 and U87 cell lines was enhanced under hypoxia compared with control group(LC3B? increased,P62 decreased,rate of GFP-LC3 puncta-positive cells was higher)in a time gradient-dependent manner.BAF treatment significantly increased LC3B-? and P62 levels of U251 and U87 cells under hypoxia.(2)MiR224-3p is down-regulated under hypoxia in GBM cell and expressed at low level in human gliomaMiRNA microarray analysis of U251 cells showed that miR224-3p was the most significantly down-regulated miRNA by hypoxia.Q-PCR results further confirmed that miR224-3p expression levels of U251 and U87 cells under hypoxia were decreased significantly.Q-PCR results of thirty cases of gliomas(14 cases of low-grade gliomas,WHO ?,? and 16 cases of high-grade gliomas,WHO ?,?)and 6 cases of normal brain tissues showed that the lower expression of miR224-3p in glioma tissues is compared with normal brain tissues.(3)MiR224-3p influences hypoxia-induced autophagy in GBM cellTransmission electron microscopy,Western blot and GFP-LC3 stable cell lines were used to detect the autophagy levels of GBM cells.In normoxia,the autophagy and autophagy flux levels of U251 and U87 cells transfected by miR224-3p inhibitors were strongly increased..Under hypoxia,overexpression of miR224-3p significantly blocked the elevation of autophagy in U251 and U87 cells.(4)MiR224-3p regulates autophagy by directly targeting both ATG5 and FIP200Among a number of predicted autophagy-related target genes of miR224-3p,overexpression and knock-down of miR224-3p apparently reduced and increased both FIP200 and ATG5 expression respectively.Moreover,co-overexpression of FIP200 and ATG5 could reverse the inhibitory effect of miR224-3p overexpression on hypoxia-induced autophagy.Further,results of dual-luciferase gene reporter assay showed that miR224-3p can directly bind to 3'UTR region of both FIP200 and ATG5.(5)MiR224-3p inversely correlates with ATG5 and FIP200 expression in human glioma tissuesThe results of immunohistochemistry showed that the expressions of HIF1 a(hypoxia marker)and LC3B(autophagy marker)in glioma tissue specimens were higher than normal brain tissue samples.The results of quantitative and statistical analysis showed that miR224-3p was negatively corre lated with the expression levels of both FIP200 and ATG5 in thirty different grade gliomas.(6)MiR224-3p suppresses glioblastoma cell proliferation and promotes hypoxia-induced apoptosisThe results of CCK-8 and EdU assays indicated that overexpression of miR224-3p substantially increased the viability and proliferation of U251 and U87 cells,verse vice.The results of Annexin V-FITC apoptosis kit and anti-cleaved-caspase3 immunocytochemistry showed that overexpression of miR224-3p significantly increased the hypoxia-induced apoptotic ratio of U251 and U87 cells.(7)MiR224-3p inhibits growth of GBM in vivoIn vivo,the glioma mouse xenograft models using U87 cells showed that there were significantly smaller volumes and lighter terminal weights of subcutaneous tumors in the miR224-3p mimic group compared with the control group.4.Conclusion(1)Through performing miRNA microarray analysis,for the first time,we confirmed that miR224-3p played a vital role in hypoxia-induced autophagy of GBM cells.(2)In GBM cells,hypoxia significantly down-regulates the expression of miR224-3p.(3)MiR224-3p inhibits autophagy activity of GBM cells by decreasing the expression of autophagy-related genes,FIP200 and ATG5.(4)MiR224-3p directly inhibited the expression of target genes by binding 3'UTR of FIP200 and ATG5 mRNA.(5)In glioma tissues,the expression of miR224-3p is lower compared to normal brain tissues;miR224-3p is negatively correlated with FIP200 and ATG5 respectively.(6)MiR224-3p suppresses proliferation and promotes hypoxia-induced apoptosis in GBM cells.(7)In vivo experiments showed that miR224-3p inhibited the growth of GBM cells.Part ?:The role and mechanism of MIF in hypoxia-induced EMT and VM formation of GBM cells1.Objective(1)To study the correlation between HIF1?(hypoxia index)with MIF and its downstream receptor CXCR4 in clinical specimens of gliomas.(2)To reveal the co-localization of HIF1?,MIF,CXCR4 and VM in clinical specimens of gliomas,and to analyze the effects of MIF,CXCR4 expression and VM positive on the prognosis of patients.(3)To clarify the influence of hypoxia on EMT and VM formation of GBM cell lines.(4)To elucidate the key role of MIF in hypoxia-induced EMT and VM formation of GBM cell.(5)Through blocking downstream CXCR4-related signaling pathway of MIF by specific drugs,to explore the mechanism of MIF in EMT and VM formation induced by hypoxia in GBM cells.(6)To investigate the effect of MIF on EMT and the downstream signaling pathway in vivo by using subcutaneous xenografts of GBM cells in nude mice.2.Method(1)Cell hypoxia treatment and detection of mRNA and protein levels After U251 and U87 cells were treated with corresponding rhMIF,AMD3100,LY294002,ISO-1 respectively under normoxia and hypoxia,the total mRNAs and proteins were extracted.The mRNAs and proteins expression levels of relevant EMT markers in 251 and U87 cells were detected by q-PCR and Western blot.(2)Cells transfectionTransient transfection of siMIF with lipofectamine 2000 was used to knock down the expression of endogenous MIF in U251 and U87 cells,and to measure the changes of expression levels of various related EMT markers.(3)Immunohistochemistry and immunofluorescence of clinical specimens25 cases of clinical glioma tissue(12 cases of low grade glioma,WHO ?,? grade and 13 cases of high grade glioma,WHO III,IV grade)and 6 Normal brain tissue were collected and then paraffin sections were prepared.The expressions of HIF1?,MIF and CXCR4 and the VM(CD34 and PAS)were assessed by immunohistochemistry.Immunohistochemistry and immunofluorescence were performed to detect the co-localization of HIF1?,MIF,CXCR4 and VM in continuous paraffin sections.(4)Cell immunofluorescenceU251 and U87 cells were treated with hypoxia and corresponding rhMIF,AMD3100,LY294002,ISO-1.Then cells were fixed by 4%paraformaldehyde,and the expression levels of MIF,CXCR4 and E-cadherin(epithelial phenotype marker)was tested by immunofluorescence.(5)VM formation of GBM cells assayU87 cells were treated with hypoxia and rhMIF,AMD3100,LY294002 and ISO-1 respectively.The effects of different treatment factors on the ability of VM formation in U87 cells were examined.(6)Nude mice experimentSubcutaneous xenograft model of nude mouse bearing human glioma tumor was established by U87 cell line.Mice with tumors were randomly divided into five groups:PBS,rhMIF,rhMIF and AMD3100,rhMIF and LY294002,ISO-1.The tumor volume was measured three weeks later.The expression levels of N-cadherin and E-cadherin were tested by immunohistochemistry.3.Results(1)The expression of MIF and CXCR4 is correlated with HIFla level in glioma specimens.The results of immunohistochemistry showed that the expression levels of MIF,CXCR4 and HIF1? were significantly higher in gliomas than in normal brain tissues,and higher grade gliomas expressed higher MIF,CXCR4 and HIF1? than those of low grade gliomas.At the same time,the statistical analysis indicated that the expression levels of MIF and CXCR4 were positively correlated with HIF1?.Immunofluorescence of continuous sections found co-localization phenomenon of MIF,CXCR4 with HIF1?in glioma tissue.(2)VM formation is correlated with the high co-expression of MIF and CXCR4 in glioma tissues.Immunohistochemistry co-staining of CD34 and PAS demonstrated that the formation of VM was apparently higher in the high grade glioma than in low grade,and the high co-expression of MIF and CXCR4 was positively correlated with the formation of VM.The results of immunohistochemistry showed that MIF,CXCR4,HIF1? and VM were co-localized in glioma tissues.Further survival analysis suggested that the survival of VM-positive glioma patients was obviously shorter than that of negative patients.(3)In GBM cells,hypoxia increases MIF and CXCR4 expressions;hypoxia and MIF induce EMT and VM formation.The results of Western blot,q-PCR and immunofluorescence showed that the expression of MIF and CXCR4 was significantly increased under hypoxia,and hypoxia and MIF promoted EMT[epithelial phenotype marker(E-cadherin)decreased and mesenchymal phenotype markers(N-cadherin,Vimentin)increased].VM formation experiments results showed that hypoxia and MIF can promote the VM formation of GBM cells.(4)Hypoxia regulates VM formation through the MIF-CXCR4-AKT-EMT pathway in GBM cells.The results of q-PCR,Western blot and VM formation indicated that MIF-induced EMT and VM formation were blocked by CXCR4 and AKT inhibitors.Moreover,hypoxia-induced EMT and VM formation can also be blocked by MIF,CXCR4 and AKT inhibitors.(5)MIF promotes the EMT of GBM cells and tumor growth in vivo.The tumor volume of MIF group was strongly higher than that of control group,MIF+ AMD3100 group and MIF+ LY294002 group.And the tumor volume of ISO-1 group was significantly smaller than that of control group.Tumor immunohistochemistry results showed that tumor cells of MIF group exhibited EMT(E-cadherin decreased,N-cadherin increased),while the other group had no significant effect.4.Conclusion(1)The VM,a compensatory mechanism to resist the treatment of anti-endothelial neovascularization,is existed in glioma tissues.(2)In glioma tissues,HIF1? is positively correlated with the expression levels of MIF and CXCR4 respectively.(3)In glioma tissues,HIF1?,MIF,CXCR4 and VM were co-localized.(4)Hypoxia can simultaneously increases the expression of MIF and CXCR4 in GBM cells and enhance the process of EMT and VM formation.(5)MIF plays a critical role in hypoxia-induced EMT and VM formation of GBMcells.(6)The elevated expression of MIF and CXCR4 in hypoxia activates the AKT pathway,promots the process of EMT,and enhances the ability of VM formation in GBM cells.(7)In vivo experiments showed that MIF promoted the malignant biology of GBM cells through CXCR4-AKT-EMT pathway.