Effects of glycosylphosphatidylinositol-anchored HDL-binding protein on glioma growth and macrophage infiltration
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摘要:目的
探讨糖基化磷脂酰肌醇锚定高密度脂蛋白结合蛋白1 (GPIHBP1)对胶质瘤生长及巨噬细胞浸润的影响。
方法首先，利用TCGA数据库分析人GPIHBP1在胶质瘤样本中的表达及巨噬细胞浸润情况，并在临床组织样本中验证上述生物信息学分析结果; 通过构建稳定过表达GPIHBP1的胶质瘤细胞系，进一步探讨GPIHBP1过表达对胶质瘤细胞增殖、凋亡、迁移和侵袭的影响。最后，通过荷瘤实验验证GPIHBP1过表达对肿瘤生长及巨噬细胞浸润的影响。
结果TCGA数据库分析显示, GPIHBP1在低级别胶质瘤中的表达高于正常组织，在高级别胶质瘤中的表达低于正常组织。此外，低级别胶质瘤中GPIHBP1的表达水平高于高级别胶质瘤，这一结果通过免疫组织化学(IHC)实验得到验证。Western blot分析验证了过表达GPIHBP1的胶质瘤细胞系构建成功。CCK-8、流式细胞术、划痕实验和Transwell实验结果显示，该稳转细胞株的增殖能力减弱，迁移能力和侵袭能力降低。荷瘤实验进一步表明，该稳转细胞株的肿瘤生长能力降低，巨噬细胞浸润减少。
结论GPIHBP1在不同分级胶质瘤中的表达差异可能与肿瘤进展相关。过表达GPIHBP1可抑制胶质瘤生长，其可能是通过影响肿瘤微环境，促进巨噬细胞向具有抗肿瘤作用的M1型极化，进而抑制胶质瘤生长。
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关键词:
- 糖基化磷脂酰肌醇锚定高密度脂蛋白结合蛋白1 /
- 胶质瘤 /
- 增殖 /
- 巨噬细胞 /
- 肿瘤微环境
Abstract:ObjectiveTo investigate the effects of glycosylphosphatidylinositol-anchored HDL-binding protein (GPIHBP1) on glioma growth and macrophage infiltration.
MethodsInitially, the expression of GPIHBP1 in glioma samples and macrophage infiltration were analyzed using TCGA database, and these bioinformatics results were validated in clinical tissue samples. A stable glioma cell line overexpressing GPIHBP1 was then established to further explore the effects of GPIHBP1 overexpression on glioma cell proliferation, apoptosis, migration, and invasion. Finally, the impact of GPIHBP1 overexpression on tumor growth and macrophage infiltration was verified through xenograft experiments.
ResultsTCGA database analysis revealed that GPIHBP1 expression was higher in low-grade gliomas compared to normal tissues, while it was lower in high-grade gliomas. Additionally, the expression level of GPIHBP1 in low-grade gliomas was higher than in high-grade gliomas, which was confirmed by immunohistochemistry (IHC). Western blot analysis confirmed the successful construction of the GPIHBP1-overexpressing glioma cell line. CCK-8, flow cytometry, scratch and Transwell assays demonstrated that the proliferation, migration and invasion capabilities of the stable cell line were reduced compared to the control group. Xenograft experiments further showed that the tumor growth and macrophage infiltration were decreased in the stable cell line.
ConclusionThe differential expression of GPIHBP1 in different grades of gliomas may be associated with tumor progression. Overexpression of GPIHBP1 can inhibit glioma growth, possibly by influencing the tumor microenvironment and promoting the polarization of macrophages towards the antitumor M1 phenotype, thereby inhibiting glioma growth.
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图 1 TCGA数据库分析胶质瘤中GPIHBP1的表达水平
A: GPIHBP1在高级别、低级别胶质瘤中与正常组织的差异; B: 胶质瘤患者生存曲线; C: GPIHBP1表达差异与免疫浸润的关系。
两者比较, **P＜0.01, ***P＜0.001。![]()
图 2 胶质瘤患者GPIHBP1表达水平及其与免疫浸润的关系
A: IHC(放大400倍)实验检测GPIHBP1在低级别胶质瘤组织临床样本中高表达，阳性结果使用红色箭头标注; B: IHC(放大200倍)实验检测IBA1在高级别胶质瘤组织临床样本中高表达。IBA1是一种小胶质细胞或巨噬细胞特异的钙结合蛋白，阳性结果使用红色箭头标注。两者比较, **P＜0.01, ***P＜0.001。
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图 3 Western blot分析过表达GPIHBP1后胶质瘤细胞中GPIHBP1的表达
GL261、U87胶质瘤细胞过表达oe GPIHBP1后检测GPIHBP1蛋白的表达, *P＜0.05, ***P＜0.001。
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图 4 CCK-8与流式细胞术检测过表达GPIHBP1时GL261与U87胶质瘤细胞增殖、凋亡情况
A、B: CCK-8与流式细胞术检测过表达GPIHBP1时, GL261与U87胶质瘤细胞增殖减少; C: 凋亡试剂盒检测过表达GPIHBP1时, GL261细胞晚期凋亡增加，总体凋亡增加; U87细胞晚期凋亡增加，总体凋亡增加。
两者比较, *P＜0.05, **P＜0.01, ***P＜0.001。![]()
图 5 细胞划痕与Transwell实验检测过表达GPIHBP1后GL261与U87胶质瘤细胞的迁移、侵袭能力
A: 细胞划痕实验显示，过表达GPIHBP1的胶质瘤细胞迁移能力减弱; B、C: Transwell实验显示，过表达GPIHBP1的胶质瘤细胞迁移、侵袭能力减弱。两者比较, **P＜0.01, **P＜0.001。
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图 6 过表达GPIHBP1对肿瘤生长及巨噬细胞浸润的影响
A: 原位荷瘤的小鼠活体成像显示，过表达GPIHBP1时肿瘤生长减慢; B: 皮下移植瘤显示，过表达GPIHBP1时肿瘤生长减慢; C: 小鼠原位荷瘤的脑片IF(放大200倍)检测Ki67与IBA1表达水平显示，过表达GPIHBP1时肿瘤增殖及巨噬细胞浸润较control少。D: 小鼠原位荷瘤的脑片IF(放大200倍)检测IBA1与INOS和ARG1共定位情况显示，过表达GPIHBP1时小胶质细胞中M1型增多, M2型减少; E: 小鼠皮下移植瘤石蜡切片IHC(放大200倍)实验显示，过表达GPIHBP1时，肿瘤增殖及巨噬细胞浸润较control少; F: 小鼠皮下移植瘤石蜡切片IF(放大200倍)实验检测F4/80与INOS和ARG1共定位情况显示，过表达GPIHBP1时巨噬细胞中M1型增多, M2型减少。F4/80是小鼠含生长因子样模体黏液样激素样受体(EMR1), 在巨噬细胞的成熟、活化过程中, F4/80蛋白表达发生显著变化。INOS: M1型小胶质细胞/巨噬细胞标志物; ARG1: M2型小胶质细胞/巨噬细胞标志物。两者比较, *P＜0.05, **P＜0.01, ***P＜0.001。
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