Molecular Mechanism Of KDELR2 In Promoting Vasculogenic Mimicry Formation In Glioma

[Objective]Glioma is the most common malignant brain tumor deriving from kinds of glial cells.Considering its’pathological characteristics of hypervascularization and highly invasion and infiltration,it can be divided into low-grade(I-II)and high-grade(III-IV)gliomas according to the WHO Classification.Vasculogenic mimicry(VM)is composed of tumor cells and matrix attached to the inner wall of the channel,which mainly occurs in highly invasive malignant tumors,and is a microcirculatory vascular-like structure with nutrition supply function.Numerous studies suggest that the existence of VM may be the primary reason for the poor efficacy of tumor anti-angiogenic targeted therapy.Therefore,the targets of ideal anti-angiogenesis therapy should be both traditional endothelial vascular and tumor cell-derived VM.We found that the VM formation in glioma is closely associated with the expression of the endoplasmic reticulum protein retention receptor 2(KDELR2).Intervening the expression of KDELR2 can regulate the ability of VM formation in glioma.Therefore,this study is aimed at exploring the function and the underlying molecular mechanisms of KDELR2 in VM formation of glioma,and attempts to provide theoretical basis and new strategies for clinical effective anti-angiogenic therapy.[Methods]1.Immunohistochemical technique was used to detect the VM of different grades of gliomas.2.Combining the bioinformatics analysis of TCGA&GEO database with RNA-sequencing transcriptome sequencing data of 34 tissues,KDELR2 was confirmed as the primary gene associated with the formation of VM in glioma.3.Furthermore,immunohistochemical technique was used to verify the close relationship between the expression of KDELR2 and the VM formation,and to analyze the relationship between the existence of VM or the level of KDELR2 expression and clinical prognosis.4.Investigate the association between the expression of KDELR2 and the abilities of VM forming in glioma cell lines.5.A glioma cell line SKMG-4 with stable knockdown of KDELR2 expression and a glioma cell line T98G with stable overexpression of KDELR2were constructed by lentivirus infection.The effects of knockdown and overexpression of KDELR2 on VM formation,cell proliferation and migration/invasion of glioma cells were studied in vitro and in vivo.6.q RT-PCR,Western blot,Co-IP and other molecular biological techniques were used to study the possible molecular mechanism of the role of KDELR2.7.Rescue experiments were used to verify the underlying molecular mechanisms,and to clarify the function of KDELR2 in promoting the VM formation in glioma.[Results]1.The expression of KDELR2 increased with the WHO grade of glioma.The survival analysis showed that the expression of KDELR2 was negatively correlated with the overall survival time of patients.2.The tumor cells around the VM structure which is CD34-/PAS+staining showed high expression of KDELR2 by CD34/PAS double staining and KDELR2 immunohistochemical staining.3.SKMG-4 glioma cell line with stable knockdown KDELR2 and T98G glioma cell line with stable overexpression of KDELR2were successfully constructed by lentivirus infection.The results of functional experiments in vitro showed that the ability of VM formation,cell proliferation and migration/invasion of glioma cells were significantly decreased after knocking down the KDELR2;The ability of VM formation,cell proliferation and migration/invasion of glioma cells were significantly enhanced after overexpression of KDELR2.The results of subcutaneous tumorigenesis in nude mice showed that the ability of tumorigenesis and the number of VM in tumor samples of SKMG-4 decreased significantly after knocking down KDELR2.4.The mechanism of KDELR2 promote the formation of VM in glioma may be up-regulating Src/PI3K/MMP2/MMP9 signal pathway by means of binding and activating Gαq.5.We used Al F4~-as Gαq activator to treat glioma cells with KDELR2 knocked down,and found that Al F4~-treatment could significantly enhance the ability of VM formation and the level of Src phosphorylation and MMP2 expression in glioma cells.[Conclusions]our results showed that the expression of KDELR2 increases along with the WHO grades of glioma,and the high expression of KDELR2 indicates the worse prognosis of patients.KDELR2 promotes the formation of VM in glioma by activating Src/PI3K/MMP2/MMP9 signal pathway with directly binding and activating Gαq.Therefore,targeting KDELR2 will help to inhibit the ability of VM formation in glioma.