摘要目的:初步探讨缺氧诱导因子(HIF)2α经miR-652-5p启动子甲基化促进胶质母细胞瘤细胞增殖、侵袭及化疗抵抗的分子病理学机制。方法:选取3个组织病理学分别为世界卫生组织(WHO)2、3、4级的原代脑胶质瘤组织标本，分别采用蛋白质免疫印迹(WB)实验和实时荧光定量反转录聚合酶链反应(RT-qPCR)检测DNA甲基转移酶1(DNMT1)和miR-652-5p的表达。取U87和A172细胞在常氧(21%O 2)与缺氧(1%O 2)环境培养12、24 h后采用RT-qPCR检测HIF2α、DNMT1、miR-652-5p的表达，采用甲基化特异性PCR(MSP)检测miR-652-5p启动子甲基化状态。与空载组相比，抑制细胞miR-652-5p表达或促进细胞miR-652-5p表达后培养48 h检测细胞侵袭，给予替莫唑胺(100 μmol/L)培养48 h后检测细胞增殖及凋亡。应用5-aza-CdR抑制细胞DNMT1表达后检测miR-652-5p表达及启动子甲基化状态。缺氧环境下培养敲除HIF2α表达的细胞48 h后，经WB实验检测DNMT1表达，采用RT-qPCR检测miR-652-5p表达，同时采用Transwell小室侵袭实验检测细胞侵袭性；予替莫唑胺(100 μmol/L)处理后，利用细胞计数试剂盒-8(CCK-8)检测细胞增殖，流式细胞术检测细胞凋亡。 结果:miR-652-5p在胶质母细胞瘤细胞(WHO 4级)中表达量低于WHO 2、3级胶质瘤( F＝135.74， P<0.001)。缺氧培养U87、A172细胞12、24 h，RT-qPCR检测提示随着缺氧时间延长，HIF2α及DNMT1表达逐渐升高，miR-652-5p表达逐渐下降(均 P<0.05)。敲除HIF2α的A172、U87细胞在予替莫唑胺处理48 h后，CCK-8检测提示增殖速率均较空载组下降(均 P<0.001)，流式细胞术检测提示其细胞早期凋亡、晚期凋亡及总体凋亡率均较空载组上升(均 P<0.05)。Transwell小室侵袭实验证实，敲除HIF2α的两组U87细胞培养48 h相比于空载组侵袭细胞数下降( F＝73.45， P<0.001)。与空载组相比，抑制miR-652-5p表达后细胞侵袭及增殖能力升高，凋亡率下降(均 P<0.05)；促进miR-652-5p表达后细胞侵袭及增殖下降，凋亡率升高(均 P<0.05)。MSP检测提示缺氧培养A172、U87细胞后miR-652-5p启动子甲基化水平上升，非甲基化水平下降；常氧培养A172、U87细胞后miR-652-5p启动子甲基化水平下降，非甲基化水平升高。DNMT1在胶质母细胞瘤细胞中表达水平高于WHO 2、3级胶质瘤( F＝64.53， P<0.001)。5-aza-CdR处理A172、U87细胞较二甲基亚砜(DMSO)对照组的DNMT1表达、miR-652-5p启动子甲基化水平均下降，miR-652-5p表达均升高(均 P<0.001)。缺氧培养敲除HIF2α表达的细胞48 h后DNMT1表达较空载组下降，miR-652-5p表达升高(均 P<0.05)。 结论:缺氧环境下HIF2α诱导miR-652-5p启动子甲基化，进而促进胶质母细胞瘤细胞侵袭、增殖及化疗抵抗。

abstractsObjective:To preliminarily investigate the molecular pathological mechanisms that hypoxia-inducible factor (HIF) 2α promotes the proliferation, invasion and chemotherapy resistance of glioblastoma cells through the methylation of miR-652-5p promoter.Methods:Three primary glioma tissue specimens with histopathological grades corresponding to World Health Organization (WHO) grades 2, 3, and 4 were selected. DNA methyltransferase 1 (DNMT1) and miR-652-5p expression levels were assessed using Western blotting (WB) and reverse transcription quantitative real-time polymerase chain reaction(RT-qPCR) analyses. U87 and A172 cells were cultured under normoxic (21%O 2) or hypoxic (1%O 2) conditions for 12 or 24 hours, after which HIF2α, DNMT1, and miR-652-5p expression levels were evaluated by RT-qPCR. Methylation-specific PCR (MSP) was employed to determine the methylation status of the miR-652-5p promoter. Following 48-hour culture with miR-652-5p inhibition or overexpression, cell invasion was analyzed; additionally, cell proliferation and apoptosis were assessed after 48-hour treatment with temozolomide (100 μmol/L). The effects of DNMT1 inhibition via 5-aza-CdR on miR-652-5p expression and promoter methylation were also examined. In a separate set of experiments, HIF2α-knockdown cells were cultured under hypoxic conditions for 48 hours, followed by WB analysis of DNMT1 expression and RT-qPCR measurement of miR-652-5p levels. Cell invasion was evaluated using Transwell assays. After temozolomide treatment (100 μmol/L), cell proliferation was measured using the Cell Counting Kit-8 (CCK-8), and apoptosis was quantified by flow cytometry. Results:The expression of miR-652-5p was significantly lower in glioblastoma cells (WHO grade 4) compared with that in lower-grade gliomas ( F=135.74, P<0.001). Under hypoxic conditions, RT-qPCR results showed that HIF2α and DNMT1 expression increased progressively over time (12 and 24 hours), while miR-652-5p expression decreased accordingly (all P<0.05). MSP analysis revealed an increase in miR-652-5p promoter methylation levels. HIF2α knockdown in A172 and U87 cells led to reduced proliferation following 48-hour temozolomide treatment as determined by CCK-8 assay (all P<0.001), and flow cytometric analysis demonstrated increased early, late, and total apoptosis compared with control groups (all P<0.05). Transwell invasion assays confirmed that HIF2α-depleted U87 cells exhibited significantly reduced invasiveness after 48-hour culture compared with controls ( F=73.45, P<0.001). Inhibition of miR-652-5p enhanced cell invasion and proliferation while reducing apoptosis (all P<0.05), whereas miR-652-5p overexpression suppressed invasion and proliferation and increased apoptosis (all P<0.05). MSP analysis further indicated that hypoxia increased methylation levels of the miR-652-5p promoter and decreased non-methylated forms, whereas normoxia had the opposite effect. DNMT1 expression was higher in glioblastoma cells than in lower-grade gliomas (WHO grade 2, 3)( F=64.53, P<0.001). Treatment of A172 and U87 cells with 5-aza-CdR significantly reduced DNMT1 expression and promoter methylation levels compared with dimethyl sulfoxide control groups, while miR-652-5p expression was markedly upregulated (all P<0.001). Finally, hypoxic culture of HIF2α-knockdown cells for 48 hours resulted in decreased DNMT1 expression and increased miR-652-5p levels compared with controls (both P<0.05). Conclusion:HIF2α promotes the proliferation, invasion and chemotherapy resistance of glioblastoma cells through the methylation of miR-652-5p promoter in hypoxia environment.