| Background and ObjectiveMeningioma is one of the most common tumors of the central nervous system, accounting for 20% of all intracranial tumors. In 2007 WHO divided meningioma into three levels according to the pathological type (WHOI-Ⅲ, malignant degree increments). In recent years, with the development of pathological technology, the proportion of WHO grade III meningiomas (malignant meningioma, MM) was significantly higher than before, accounting for 15%-20% of all meningiomas Meningioma pathological grading is still the main reason affects the prognosis [1-5]. Comprehensive surgical treatment is the main method for therapy [6]. however, after the comprehensive treatment of malignant meningioma, the overall recurrence rate is as high as 80%,5 year survival rate is almost 0. Its prognosis is similar with glioblastoma’s which is the most malignant intracranial tumor [7]. Its high recurrence rate and mortality rate make it become one intracranial tumor of the most difficult to overcome. Looking for new treatments and methods are crucial to the prevention of malignant meningioma and the improvement of prognosis.Malignant meningioma is the tumor extremely rich in the blood supply. On the one hand, the tumor vascular network system ensures the tumor cells with rich oxygen supply, nutrient and promotes metabolite excretion and tumor growth; on the other hand,the incomplete vascular wall structure in tumor tissue and the variable thickness of the basement membrane shall be conducive to tumor cells into the lumen of blood vessel. Vascular endothelial cell growth factor (VEGF) is considered to be the most important factor in regulating angiogenesis of tumor, which’s binding to its receptor has activated tyrosine kinase receptor and promoted vascular endothelial cell proliferation, angiogenesis. VEGF expressed in 60% of meningioma, almost 100% expressed in malignant meningiomas [8]. Bevacizumab, a humanized monoclonal antibody against VEGF, which binds to the VEGF receptor competitively with the endogenous VEGF, inhibits the endothelial cell mitosis, reduces neovascularization and blocks the supply of blood, oxygen and other nutrients required for tumor growth. The bevacizumab has become a new treatment of malignant meningioma [8]. The safety of malignant meningioma treatment has been recognized. However, the targeted therapy of VEGF is often concerning the search of a new targets for anti angiogenesis therapy or a new combination of targeted therapy, which has important significance in prolonging the overall survival of patients with malignant meningiomas and improving the quality of life.In 1999, American scholar Maniotis in melanoma found a no endothelial cells involved in the microcirculation of the pipeline called vasculogenicmimicry (VM) [8]. Tumor cells mimicking body vascular formation, proposed pipeline network like structure with the original.VM blood vessel connected to endothelium-dependent is proposed the only way to challenge the traditional concept of tumor microcirculation, but also for the theory of tumor angiogenesis which is an important supplement of.VM helps explain some malignant tumor invasion, metastasis and biological behavior of high resistance to anti angiogenic therapy. Compared with the classical tumor angiogenesis, VM tube wall is mainly composed of a layer of PAS positive basement membrane enclosed. The outer lining to tumor cells but not endothelial cells, VM within the lumen of plasma and red blood cell flow. The outside of the tube wall lining of tumor cells in the basal release of proteolytic enzymes dissolved after the film is directly into the blood, which is more conducive to the invasion and metastasis of the tumor. In many malignant tumors found in the survival and prognosis of patients with VM are closely related to the.Deep understanding of the phenomenon and mechanism of VM, the tumor diagnosis, clinical value prediction and treatment will be gradually reflected.VM pathogenesis is still not clear, relevant research mainly concentrated in tumor cells differentiation, vasculogenic mimicry structure characteristics and tumor micro environment. Involved in the formation of tumor cells of the VM simulation function and phenotype of endothelial cells, and endothelial cells are mesenchymal cells of a and in VM formation in the process of epithelial tumor cells to qualitative endothelial cell transdifferentiation (EMT) play an important role.Since VM is an angiogenesis mode that is independent in VEGF in malignant tumor. We envision bevacizumab as the treatment of malignant meningioma,which is not very ideal, in malignant meningiomas may exist in a non dependent VEGF angiogenesis. So we on 12 cases of malignant meningioma were immunohistochemical staining. Results found, in 6 cases (50%) malignant meningioma existing VM phenomenon. However, what is the cause of malignant meningioma in VM formation? Why VM exists only in malignant meningiomas.1p36 loss of heterozygosity (LOH), is the most significant adverse prognosis of malignant meningioma. The molecular events occurred in 70-100% malignant meningioma [13-15]. many 1p chromosome genes, including CDKN2C, RAD54L, EPB41, GADD45A, RAD54L, and ALPL, were studied in malignant meningiomas, but found no abnormalities can explain the biological behavior of malignant meningioma and the poor prognosis of [16,17]. The mechanism that 1p36 led to poor prognosis of malignant meningioma, is worthy of further research. This research advance on 30 patients with malignant meningioma by iFISH 1p36 detection of loss of heterozygosity,19 cases of VM were found the phenomenon of malignant meningioma in 1p36 were deficiency, and the other 11 cases without VM 1p36 the phenomenon of malignant meningioma in 19 cases. We found 19 cases with deletion of 1p36 in malignant meningiomas (LOH-MM) and 11 cases of malignant meningiomas without deletion of 1p36 (NLOH-MM) though miRNA microarray analysis. The results showed that the expression of more than 41 miRNAs is two times higher than LOH-MM, there are 26 miRNAs expression by two times; the miR-200a in LOH-MM is down more than 20 times.MiR-200a as a member of miR-200 family, most malignant tumors for expression of fine cut state, and regulating its downstream target gene expression of [18-21]. In our literature and through bioinformatics analysis, miR-200a can directly hit the target-catenin mRNA (beta CTNNB1 transcription), down-regulation of miR-200a may activate the classical Wnt pathway and EMT induced effects of tumor occurrence and development. In order to verify this assumption is not in the presence of LOH-MM, we use immunohistochemical techniques such as western, found that compared with NLOH-MM, Wnt the LOH-MM pathway activated, mainly for beta-catenin transfer to the cytoplasm and nucleus; at the same time some EMT markers, such as a significant down-regulation of E-cadherin in LOH-MM, and N-cadherin Expression was significantly upregulated. On the basis of preliminary work, we infer that the VM phenomenon in malignant meningiomas occur and 1p36 deletion resulted in EMT related 1p36 deletion, through down-regulation of mir-200a expression, activation of the canonical Wnt signaling pathway, EMT induced, causing the formation of VM, which leads to bevacizumab monoclonal antibody targeting the VEGF to anti angiogenesis therapy invalid, and cause tumor proliferation and invasion ability strengthened, leading to poor prognosis. While at the same time targeting the vascular endothelial growth factor (VEGF) and Wnt pathways to treatment may have potential clinical significance for the treatment of malignant meningioma.If this study carried out smoothly, we will provide the theoretical basis of regarding the 1p36 deletion as a molecular event of poor prognosis in malignant meningioma, and also provide new targets and combination therapy for the comprehensive treatment of malignant meningioma which has important significance to improve the prognosis of patients and reduce the postoperative recurrence rateContent and Methods1.Study on the correlation between Ip36 deletion and miR-200a down in different grades of meningiomas.60 cases of meningioma patients were included in the study who had been diagnosed by pathology departmen of Nanfang Hospital Affiliated to Southern Medical University,collected from December 2013,4 to 2015,12 in Department of Neurosurgery including 30 cases of WHO grade I meningioma,20 cases of WHO grade Ⅱ meningioma,10 cases of WHO grade III meningioma.We have complete data of all the above cases that before surgery and the clinical data and imaging mode. The experimental methods:using site-specific (LSI) Ip36 probe fluorescence in situ hybridization (FISH) detect the malignant meningioma tissues (WHO Ⅱ-Ⅲ) and ordinary tumor tissues (WHO I) respectively at 1p36 locus deletion; fluorescence quantitative detection of miR-200a two kinds of cells and normal arachnoid, statistical analysis of the expression of miR-200a in each group,and analyzing the correlation between miR-200a and 1p36 deletion.2. Preliminary exploration of β-catenin protein expression level and vasculogenicmimicry (VM) in meningioma10 cases of paraffin tissue specimens in each grade of meningioma were enrolled in this study, using immunohistochemical method to detect β-catenin nuclear transfer and statistics; periodic acid Schiff reaction (PAS) and CD31 double staining technique was used to detect the level meningioma VM occurrence rate and statistics; take the three types of meningioma primary cells to do the three-dimensional cell culture, established VM model and observed the angiogenic capacity of meningioma.3. The activation of Wnt/ -catenin signaling pathway induced by the downregulation of miR-200a could promote malignant meningioma cell proliferation, migration and angiogenesis.Establish primary cell culture model of malignant meningiomas (WHO grade III) and verify it. Through lentivirus transfection regulated miR-200a level in meningioma cell, as overexpression group (miR-200a+) and down regulation group (miR-200a-), set the negative control group (negative control), using Western blot to detect β-catenin protein and EMT proteins (ZEB1, Twist 1, E-cadherin, vimentin). β-catenin nuclear transfer were observed by immunofluorescence r, the ability of cell proliferation was detected by CCK-8 cell viability assay, cell apoptosis, cell cycle) and Cell migration and invasion were detected bycell scratch test, Transwell assay. And tumorigenicity was detected by nude mice subcutaneous tumorigenicity; the ability of angiogenesis were assayed by three-dimensional cell culture.4 statistical methodsStatistical analysis was performed using SPSS 19 software. Measurement data were expressed by X+s, were analyzed using two independent samples t test. P<0.05 has significant difference.Results1.Study on the correlation between 1p36 deletion and miR-200a down in different grades of meningiomas.Using FISH probe hybridization technique for the detection of different levels of 1p36 deletion in meningiomas. The results showed that 30 cases of grade I meningiomas had no loss of 1p36, the 1p36 loss were mainly in the WHOII-III meningiomas,and the loss rate was 63.3%(19/30). Using QRT-PCR to detect mir-200a down level. The results show that the WHO grade I meningiomas cut about 2-3 times, LOH-MM (1p36 deletion of malignant meningiomas) down regulation cut about 26-37 times, NLOH-MM (no Ip36 deletion of malignant meningiomas) reduction ratio of 3-5 times. The results show that the 1p36 deletion and mir-200a down mainly exist in malignant meningiomas, and 1p36 deletion and mir-200a down-regulation correlated significantly (P< 0.05).2. Preliminary exploration of β-catenin protein expression level and vasculogenicmimicry (VM) in meningioma.Immunohistochemical staining was used to observe the expression level of β-Catenin protein. The results showed that β-catenin protein occured cytoplasmic transfer and nuclear transfer as more as the increase of tumor pathology level. Using periodic acid Schiff reaction (PAS) and CD31 double staining found that VM rate is about 15%, which grade I meningiomas found no VM phenomenon in malignant meningioma. Primary meningioma cells in three-dimensional culture results showed that the WHO grade I meningiomas is difficult to form VM, and malignant meningiomas (who II-III) easily formed typical VM structure. This result is consistent with the one of PAS-CD31 double-staining.3 The activation of Wnt/p-catenin signaling pathway induced by the downregulation of miR-200a could promote malignant meningioma cell proliferation, migration and angiogenesis.When Lentiviral transfection was assayed successfully, the expression level of EMT and VM related protein were assayed by Western blot method, results showed that (miR-200a-) cells, β-catenin, twist 1, VE-cadherin protein expression increased, E-cadherin expression decreased, while the result of the (miR-200a+) group were on the contrary. The downregulation of miR-200a could activate Wnt/β-catenin signaling pathway, triggered EMT process, VE-cadherin (VM associated protein) changes indicating that down-regulation of the miR-200a may also be indirect factors which affect the formation of VM cells. Immunofluorescence results showed that β-catenin protein was found obviously in cell nuclear transfer in the (miR-200a-) group, and the (miR-200a+) group did not appear, indicating that down-regulation of the miR-200a can be targeted activation of β-catenin protein at the same time. Though the proliferation and invasion assay we found the cells of the (miR-200a-) group have stronger cell viability, proliferation, Invasion and anti apoptotic ability compared with the cells of the (miR-200a+) group.In the assay of tumorigenicity in nude mice,the tumor volume formed by the (miR-200a-) group were larger. Cells after transfection with three-dimensional culture also indicated that the (miR-200a-) group can form a typical VM, and (miR-200a+) group not easily formed.ConclusionVM phenomenon occurred in malignant meningiomas may be related to EMT process caused by 1p36 deletion, while 1p36 deletion can result in the down-regulation of miR-200a, and then activate the canonical Wnt pathway along with the EMT and the formation of VM, which leads to invalid on bevacizumab VEGF targeted therapy of anti angiogenesis, and strengthen the proliferation and metastasis of tumor, ultimately leading to poor prognosis. Both target the VEGF and Wnt pathway may have a better therapeutic effectiveness in clinical treatment of malignant meningioma. The results of this study provides a theoretical basis for verifying deletion of 1p36 as a molecular event cause poor prognosis of malignant meningioma and also provide new targets and combination therapy for the comprehensive treatment of malignant meningioma which has important significance to improve the prognosis of patients and reduce the postoperative recurrence rate... |
PDF Full Text Request