Function And Biological Significance Of Aquaporin1on Tumor Associated Microglia

Background introductionGlioma is the most common malignant tumor of the central nervous system,accounting for about 80% of intracranial malignant tumors.Because of its aggressive,short recurrence time,poor prognosis,a serious threat to the patient’s survival time and quality.In recent years,a large number of studies have revealed the molecular classification of malignant glioma,tumorigenesis,invasion,recurrence and part of the mechanism of resistance.However,as heterogeneous tumors,microenvironment of glioma including neural stem cells,microglia,endothelial cells and other microenvironmental cells and tumor cell interaction is also an important cause of tumorigenesis,invasion and recurrence.The macrophages in the tumor microenvironment are called Tumor associated microglia(TAMs).TAMs that are infiltrated into the tumor are regulated by tumor cells and microenvironment.TAMs also secrete various cytokines and chemokines are involved in tumor remodeling,immunosuppression and angiogenesis,which are closely linked to tumor growth,invasion,and tolerance to chemoradiotherapy.A number of studies have shown that TAMs in glioma tissue are the most important inflammatory cells(30% of tumor tissue).The degree of infiltration is closely related to the classification and prognosis of gliomas,indicating that TAMs has played an important role in promoting the development.TAMs in gliomas have potent plasticity and diversity,and are activated under different cytokines,inflammatory factors and chemokines,showing different polarities.Studies have shown that TAMs in malignant gliomas promote tumor invasion and angiogenesis by expressing classical M2 markers such as TGF-β,MT1-MMP,MMP9,VEGF,and Arg1.Therefore many researchers define TAMs as M2-type microglia(alternatively activated microglia).Exploring the mechanism of malignant glioma regulation of TAMs polarization,and finding the target of reversing its tumor phenotype and related drugs,may be a new method of adjuvant treatment of malignant glioma.However,the mechanism of TAMs polarization in gliomas is not entirely clear,and the key molecules require further study and discovery.Aquaporin 1(AQP1)is a small hydrophobic transmembrane channel that plays a leading role in the transmembrane transportation of water molecules.It has been suggested that the role of AQP1 is just to facilitate transport of water.Recent studies have found that the expression of AQP1 is closely related to the clinical characteristics of many types of tumors,and may even be an independent predictor of certain tumors such as colorectal cancer prognosis.In the case of intracranial gliomas,AQP1 was found to be highly expressed in gliomas,and it was interesting to note that its expression in the surrounding was significantly higher than in the core.AQP1 is involved in cells not only tumor cells but also microenvironmental cell migration,angiogenesis,morphological changes.Studies have confirmed that AQP1 is involved in the polarization of macrophages,and for microglia cells with strong deformability,their function is found to be directly regulated by morphological changes.So whether AQP1 regulate the polarization and migration of microglia,and therefore have an effect on the function of TAMs to promote tumor development? The aim of this study was to investigate the function of AQP1 on microglia,and to explore its role in the development of glioma.Method Part I: Isolate primary microglia;synthesize and transfected AQP1 siRNA into microglia cell line BV2 to identify the efficiency.Part II: first compare the wild type and AQP1 knockout type microglia morphology;(Arg1,IL6,IGF-1,CD206,etc.),protein(Arg1,iNOS)were detected by real-time PCR and Western blotting.The isolated microglia and AQP1 knockout microglia were detected by Western blotting to analyze the polarization regulation of AQP1 on microglia.PartIII: the expression of AQP1 in TAMs of gliomas was analyzed by immunofluorescence of IBA1 and AQP1 in human normal brain tissue and human glioma specimens.Then,the mouse GL261 tumor model was constructed,and the expression of AQP1 in TAMs of gliomas was analyzed by immunofluorescence with IBA1 and AQP1.The expression of AQP1 in TAMs was confirmed by Western Blotting,and the expression of AQP1,AQP3,AQP4 and AQP9 was detected by Real-time PCR.The glioma cell lines of GL261 were stimulated with the conditioned medium of wild type and AQP1 knockout microglia.The changes of their migration ability and proliferative ability were measured and the changes of AQP1 and MMP9 were detected by Western Blotting.Additionally,identify the significance of AQP1 changes in gliomas by using siRNA on GL261.Finally,the survival time and in vivo experiment of GL261 glioma model of wild type and AQP1 knockout mice were mearured to confirm in vitro experiments.Result1.The transfection efficiency of AQP1 siRNA was high,and the fluorescence intensity of 50 nmol and 75 nmol AQP1 siRNA group was significantly lower than that of negative control group.Western Blotting result identified a significant decrease in AQP1 expression in the 50 nmol siRNA group and the 75 nmol siRNA group.2.Morphology of AQP1 knockout microglia cells and IL4 stimulation group of microglia(M2 type microglia)are similar to the performance of long synaptic morphology.When silencing BV2 cells with 50 nmol AQP siRNA,Transwell experiments and scratche assays showed that the migration ability was significantly improved.Real-time PCR analysis of wild-type and AQP1 knock-out microglia polarization-related genes showed that compared with wild-type,AQP1 knock-out microglia expressed more M2 markers(Arg1,CD206,MMP9),fewer M1(iNOS,IL6)markers and minimal AQP1 and VEGF.AQP siRNA silenced BV2 cells,and Western Blotting found that iNOS expression decreased significantly.3.Compared with microglia(IBA1 positive cells)in human normal brain tissue,the fluorescence intensity of AQP1 on TAMs in glioma was significantly decreased.The fluorescence intensity of AQP1 on TAMs in GL261 glioma model was significantly lower than that in normal brain tissue(P <0.05).AQP1 knockdown of microglia conditioned medium(AQP1KO CM)can significantly increase glioma cells migration compared to stimulation of wild-type microglia conditioned medium(WTCM).Both WTCM and AQP1 KOCM could increase the proliferation of glioma cells,but the difference between AQP1 KOCM and WTCM was not significant.The expression of AQP1 and MMP9 was up-regulated by glioma stimulated by 50 nm and 75 nmol AQP1 siRNA microglia conditioned medium(50 nM siCM and 75 nM siCM).Silencing AQP1 on GL261 glioma cells can significantly reduce the ability of glioma cells to migrate.The mean survival time of AQP1 knockout tumor bearing mice was shorter than that of wild type tumor bearing mice,but the difference between the two groups was not statistically significant(P = 0.0874).Conclusion: AQP1 defiency can cause microglia polarization towards M2 phenotype and increase migration ability.Glioma cells induced AQP1 deficency on TAMs and in turn prove invasion and progressioin of tumor.