HIF-1α促进恶性脑膜瘤血管发生的分子机制  

Molecular Mechanisms of HIF-1αon Angiogenesis in Malignant Meningioma

作　　者：帕热哈提江·依孜木 麦伍兰江·阿卜杜热西提 阿布都克尤木·阿布都吉力力 Parehatijiang Yizimu;Maiwulanjiang Abudurexiti;Abudukryumu Abudujilili(Department of Neurosurgery,People’s Hospital of Xinjiang Uygur Autonomous Region,Urumqi,830001,China)

机构地区：[1]新疆维吾尔自治区人民医院神经外科,乌鲁木齐市830001

出　　处：《医学分子生物学杂志》2023年第5期390-396,共7页Journal of Medical Molecular Biology

基　　金：新疆维吾尔自治区自然科学基金(No.2019D01C167)。

摘　　要：目的探讨HIF-1α在恶性脑膜瘤血管发生中的关键作用机制。方法qPCR法和免疫组织化学法测定正常胎盘组织和良性(WHOⅠ级)至复发性(WHOⅡ~Ⅲ级)脑膜瘤组织中HIF-1α和IGF1R的mRNA和蛋白质表达水平。双荧光素酶报告基因分析和ChIP实验证明HIF-1α与IGF1基因调控区的直接作用关系。采用shRNA敲低间变性脑膜瘤CRL-3370细胞中HIF-1α的表达。ELISA法测定敲低HIF-1α后CRL-3370细胞分泌IGF1和VEGF的水平变化。蛋白免疫印迹法测定细胞中p-IGF1Rβ、IGF1Rβ和VEGFR2的表达水平变化。建立CRL-3370细胞与HUVEC2的共培养体系,通过缺氧培养诱导CRL-3370细胞中HIF-1α表达并随后进行shRNA敲低。CCK-8法测定共培养体系中HUVEC2的细胞增殖能力;qPCR法测定其胞内VEGF、ANGP1和MMP2 mRNA的表达水平;划痕实验和Transwell细胞侵袭实验测定细胞迁移和侵袭能力。结果随着脑膜瘤由良性(WHOⅠ级)至复发性(WHOⅡ~Ⅲ级)进展,脑膜瘤组织中HIF-1α和IGF1R的mRNA和蛋白质表达水平增强(P<0.01)。与对照组相比,IGF1组的荧光素酶活性明显上调;与IGF1组相比,删除位点4组的荧光素酶活性被明显抑制(P<0.05)。ChIP实验结果显示,HIF-1α组IGF1site-4的DNA含量明显高于其对应IgG组的DNA含量(P<0.05)。与shRNA NT组相比,HIF-1αshRNA组CRL-3370细胞分泌IGF1和VEGF的水平明显降低;胞内p-IGF1Rβ、IGF1Rβ和VEGFR2的表达水平明显降低。共培养体系中HUVEC2的增殖能力明显降低;其胞内VEGF、ANGP1、MMP2 mRNA表达水平明显降低;细胞迁移和侵袭能力明显降低(P<0.05)。结论复发性脑膜瘤上调HIF-1α直接作用并增强IGF1/IGF1R轴促进HUVEC2的血管发生和肿瘤恶性进展。Objective To investigate the key mechanisms of HIF-1αon the Angiogenesis in malignant meningioma.Methods The mRNA and protein expression levels of HIF-1αand IGF1R in normal placenta tissues,benign meningioma tissues(WHO gradeⅠ)and recurrent meningioma tissues(WHO gradeⅡ-Ⅲ)were determined by qPCR and immunohistochemistry.Dual-luciferase gene reporter assay and ChIP assay were used to verify the direct interaction between HIF-1αand IGF1 gene regulatory region.Knockdown of HIF-1αexpression in anaplastic meningioma CRL-3370 cells were performed by using HIF-1αshRNA.ELISA was used to determine the secretion of IGF1 and VEGF in the CRL-3370 cells after HIF-1αknock-down.Western blotting was used to determine the expression levels of p-IGF1Rβ,IGF1Rβand VEGFR2 in CRL-3370 cells.A co-cultured system of CRL-3370 cells and HUVEC2 was established,and the expression of HIF-1αin CRL-3370 cells was induced by hypoxia condition and followed by the shRNA knocking-down.CCK-8 assay was used to determine the cell proliferation ability of HUVEC2.qPCR assay was used to determine the VEGF,ANGP1 and MMP2 mRNA expression levels in HUVEC2 cells.Wound-healing assay and transwell assay were used to determine the migration and invasion ability of HUVEC2 cells.Results The mRNA and protein expression levels of HIF-1αand IGF1R in meningioma tissues increased as the meningioma progressed from benign(WHO gradeⅠ)to reccurrent(WHO gradeⅡ-Ⅲ)(P<0.01).The luciferase activity in the IGF1 group was significantly up-regulated when compared with that in the Control group.The luciferase activity in the deletion site-4 group was significantly inhibited when compared with that in the IGF1 group(P<0.05).ChIP experiment showed that the DNA content of site-4 in the HIF-1αgroup was significantly higher than that in the corresponding IgG group(P<0.05).The levels of secreted IGF1 and VEGF in the HIF-1αshRNA group were significantly reduced when compared with those in the shRNA NT group.The intracellular expression levels of p-IGF1Rβ,IGF1Rβ

关 键 词：脑膜瘤 HIF-1Α IGF1/IGF1R轴 血管发生 

分 类 号：R739.9[医药卫生—肿瘤]