| Objective:Meningioma is one of the most common tumors in the central nervous system,accounting for more than 36%of the tumors.The World Health Organization(WHO)classifies meningiomas into three histopathological grades.Most meningiomas are WHO grade 1 with low growth rate,but 10%to 12%meningiomas are atypical or anaplastic.Due to the rarity of malignant meningioma,the clinical studies are lack and most studies are based on small sample size.Therefore,the clinical characteristics,treatment and prognosis of malignant meningiomas are still not clear.More importantly,malignant meningiomas grow rapidly with median survival time of only 2-3 years.Surgery is currently the maj or treatment,but the tumor recurrent rate is still high after surgical resection.Champeux et al.found that the time interval between the first operation and second operation for malignant meningioma was only 1.3 years.Hence,treatment of malignant meningioma is still a huge challenge for neurosurgeons.In addition,all kinds of cytotoxic drugs,including targeted drugs,have not shown significant efficacy,and there is still no systemic therapy available for malignant meningioma.Therefore,both studying the genes change and signal transduction pathways that driving the progression and recurrence of meningioma,and searching the effective molecular therapeutic targets has become an important direction in basic research of meningioma.The malignant biological behavior of malignant meningioma mainly includes abnormal proliferation of tumor cells,enhanced neovascularization,enhanced invasiveness of cells and inhibition of apoptosis.Serpinf 1 gene is an important antitumor factor,which plays anti-tumor role through various pathways,including inhibiting tumor angiogenesis,or directly inhibiting tumor cell proliferation,invasion and metastasis,and promoting tumor cell apoptosis.Serpinf 1 is a classic tumor suppressor involved in a variety of cancers,including prostate,ovarian,pancreatic,melanoma.However,the role of Serpinf 1 gene in the development,progression and prognosis of malignant meningioma is still unclear and no relevant studies have been conducted.Hence,firstly,this study aimed to explore the clinical characteristics and prognostic factors of malignant meningioma based on single-center clinical data.In addition,we used the meningioma tissue samples,malignant meningioma cell line(IOMM-Lee)and HUVEC cell line to explore the mechanisms of Serpinf 1 gene and its coding protein(PEDF)in the progression of meningioma.Materials and Methods:1.The institutional clinical study:we collected the clinical and follow-up data of patients with malignant meningioma who underwent surgical resection at department of neurosurgery in West China Hospital from January 2009 to July 2020.Cox univariate and multivariate analyses were used to analyze the associations among clinical features and outcome,such as patient age,sex,time of onset,radiological features,preoperative radiotherapy,the extent of surgical resection and postoperative radiotherapy.2.Differential expression of clinical samples:The expression level of Serpinf 1 gene in meningioma tissues and normal brain tissues was detected by RT-qPCR.We further used immunohistochemical analysis to detect the PEDF protein(encoded by Serpinf 1 gene)in different grades of meningiomas and explored the relationship between the PEDF protein and tumor recurrence rate in high grade meningioma.3.Study on the biological function of Serpinf 1 gene in malignant meningioma:based on the lentiviral vector system,we established a stable transgenic IOMM-Lee OVserpinf 1cell line to determine the effect of Serpinf 1 gene on the proliferation,migration,invasion and apoptosis in IOMM-Lee and HUVEC cells.We further explored the effect of Serpinf 1 gene in vivo based on orthotopic and subcutaneous malignant meningiomas models in nude mice.4.Exploring the mechanism of Serpinf 1 gene in inhibiting the progression of malignant meningioma:based on the transcriptome sequencing of IOMM-LeeGFP and IOMM-Lee OV-serpinf 1 and also the literature results,we explore the possible mechanisms of Serpinf 1 gene in suppressing the growth of malignant meningioma.Results:1.An institutional clinical study:70 patients with malignant meningioma were included in this study.The mean age was 52.97± 15.38 years,and the most common clinical symptom was headache(57.1%).The median follow-up time was 14.5 months,and 43(61.4%)patients had tumor recurrence.Multivariate Cox regression analysis showed that patients underwent total tumor resection(HR:2.928,P=0.003,95%confidence interval:1.425-6.016)and postoperative radiotherapy(HR:2.035,P=0.041,95%confidence interval:1.029-4.023)had significantly longer progression-free survival.There were 39(55.7%)patients died eventually,and multivariate Cox regression analysis showed that patients underwent total tumor resection(HR:3.016,P=0.006,95%CI:1.369-6.648)and postoperative radiotherapy(HR:4.067,P<0.001,95%CI:1.934-8.551)had significantly longer overall survival time.2.Differential expression of Serpinf 1 gene in clinical samples:RT-qPCR showed that the expression level of Serpinf 1 gene in normal dura and WHO grade 1 meningiomas was significantly higher than that in high-grade meningiomas(WHO grade 2).Immunohistochemistry showed the expression of PEDF protein was significantly decreased in high-grade meningiomas.Moreover,in high-grade meningiomas,the recurrence rate in PEDF protein negative group was significantly higher than that in positive group(P=0.024).3.Study on the biological function of Serpinf 1 gene in malignant meningioma:The low expression of Serpinf 1 gene and its coding protein PEDF in malignant meningioma IOMM-Lee cell line was confirmed by qPCR,WB and immunofluorescence methods.We used lentiviral vector to establish stable IOMM-Lee OV-Serpinf 1 cell lines that overexpressing Serpinf 1 gene,and we confirmed the cell lines using the methods of RT-qPCR,WB and immunofluorescence.We found that Serpinf 1 overexpression could inhibit the proliferation and migration of IOMM-Lee cells,but had no significant effect on apoptosis and invasion.Previous studies have suggested that PEDF can play the antitumor role by inhibiting angiogenesis,and PEDF is a secretory protein.Next,we used IOMM-LeeGFP and IOMM-LeeOV-Serpinf1 conditioned medium to intervene the Umbilical vein endothelial cells(HUVEC);and found that IOMM-LeeOV-Serpinf1 conditioned medium inhibited HUVEC proliferation,migration,and tube formation.The subcutaneous and brain models of nude mice showed that the tumor growth rate in Serpinf 1 overexpression group was significantly slower than that of the control group.Immunohistochemistry of subcutaneous tumors showed that the expression level of PEDF was significantly increased,and the microvascular density(MVD)was significantly decreased in the Serpinf 1 overexpression group.4.Exploring the mechanism of Serpinf 1 gene in inhibiting the progression of malignant meningioma:we combined the transcriptome sequencing results and the literature reported proteins,and validated the expression of Wnt 3A,β-catenin,HIF-1α,NF-kB,pNF-kB,PI3K,pPI3K,VEGF,mTOR,p-mTOR and P53 in these two different cell lines.We found that pPI3K,pmTOR,VEGF,pNF-κ B were significantly decreased in the Serpinf 1 overexpression group,and the residual protein level was not significantly changed.Conclusion:1.The prognosis of malignant meningioma is poor.Total resection and postoperative radiotherapy are the most important factors affecting the prognosis.The invasion of bone,brain tissue and venous sinus,and Ki67 index are not related to the prognosis.2.The expression level of Serpinf 1 gene in WHO grade 1 meningiomas was significantly higher than that in high grade meningiomas.PEDF protein expression was significantly decreased in high-grade meningiomas,and was negatively correlated with tumor recurrence.3.Overexpression of Serpinf 1 could inhibit the proliferation and migration of IOMM-Lee cells,but had no significant effects on invasion and apoptosis.Serpinf 1 overexpressed conditioned medium significantly inhibited the proliferation,migration and lumen formation of HUVEC cells.The antitumor effect of Serpinf 1 gene was further confirmed in vivo animal experiments.4.Serpinf 1 gene may inhibit IOMM-Lee cell proliferation and migration by inhibiting PI3K/mTOR and NF-κB signaling pathways;and the Serpinf 1 gene may inhibit angiogenesis by inhibiting tumor vascular endothelial cell proliferation,migration,tube formation and down-regulation of VEGF expression. |
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