CLEC19A 过表达抑制胶质母细胞瘤中肿瘤细胞的增殖/迁移，并促进细胞凋亡，同时抑制 PI3K/AKT/NF-κB 信号通路。

BACKGROUND: GBM is the most frequent malignant primary brain tumor in humans. The CLEC19A is a member of the C-type lectin family, which has a high expression in brain tissue. Herein, we sought to carry out an in-depth analysis to pinpoint the role of CLEC19A expression in GBM. METHODS: To determine the localization of CLEC19A, this protein was detected using Western blot, Immunocytochemistry/Immunofluorescence, and confocal microscopy imaging. CLEC19A expression in glioma cells and tissues was evaluated by qRT-PCR. Cell viability, proliferation, migration, and apoptosis were examined through MTT assay, CFSE assay, colony formation, wound healing assay, transwell test, and flow cytometry respectively after CLEC19A overexpression. The effect of CLEC19A overexpression on the PI3K/AKT/NF-κB signaling pathway was investigated using Western blot. An in vivo experiment substantiated the in vitro results using the glioblastoma rat models. RESULTS: Our in-silico analysis using TCGA data and measuring CLEC19A expression level by qRT-PCR determined significantly lower expression of CLEC19A in human glioma tissues compared to healthy brain tissues. By employment of ICC/IF, confocal microscopy imaging, and Western blot we could show that CLEC19A is plausibly a secreted protein. Results obtained from several in vitro readouts showed that CLEC19A overexpression in U87 and C6 glioma cell lines is associated with the inhibition of cell proliferation, viability, and migration. Further, qRT-PCR and Western blot analysis showed CLEC19A overexpression could reduce the expression levels of PI3K, VEGFα, MMP2, and NF-κB and increase PTEN, TIMP3, RECK, and PDCD4 expression levels in glioma cell lines. Furthermore, flow cytometry results revealed that CLEC19A overexpression was associated with significant cell cycle arrest and promotion of apoptosis in glioma cell lines. Interestingly, using a glioma rat model we could substantiate that CLEC19A overexpression suppresses glioma tumor growth. CONCLUSIONS: To our knowledge, this is the first report providing in-silico, molecular, cellular, and in vivo evidences on the role of CLEC19A as a putative tumor suppressor gene in GBM. These results enhance our understanding of the role of CLEC19A in glioma and warrant further exploration of CLEC19A as a potential therapeutic target for GBM.

背景：GBM 是人类最常见的恶性原发性脑肿瘤。CLEC19A 是 C 型凝集素家族的成员，在脑组织中高表达。在此，我们试图进行深入分析，以确定 CLEC19A 表达在 GBM 中的作用。

方法：为了确定 CLEC19A 的定位，使用 Western blot、免疫细胞化学/免疫荧光和共聚焦显微镜成像检测该蛋白。通过 qRT-PCR 评估神经胶质瘤细胞和组织中的 CLEC19A 表达。通过 MTT 测定、CFSE 测定、集落形成测定、划痕愈合测定、Transwell 试验和流式细胞术分别检测 CLEC19A 过表达后细胞活力、增殖、迁移和凋亡。使用 Western blot 研究 CLEC19A 过表达对 PI3K/AKT/NF-κB 信号通路的影响。使用胶质细胞瘤大鼠模型的体内实验证实了体外实验结果。

结果：通过 TCGA 数据的计算机分析和 qRT-PCR 测量 CLEC19A 表达水平，我们发现与健康脑组织相比，人胶质肿瘤组织中 CLEC19A 的表达明显较低。通过 ICC/IF、共聚焦显微镜成像和 Western blot，我们可以证明 CLEC19A 可能是一种分泌蛋白。几项体外检测结果表明，在 U87 和 C6 神经胶质瘤细胞系中过表达 CLEC19A 与细胞增殖、活力和迁移的抑制有关。此外，qRT-PCR 和 Western blot 分析表明，CLEC19A 过表达可降低神经胶质瘤细胞系中 PI3K、VEGFα、MMP2 和 NF-κB 的表达水平，增加 PTEN、TIMP3、RECK 和 PDCD4 的表达水平。此外，流式细胞术结果表明，CLEC19A 过表达与神经胶质瘤细胞系中显著的细胞周期阻滞和促进细胞凋亡有关。有趣的是，使用胶质细胞瘤大鼠模型，我们可以证实过表达 CLEC19A 可抑制胶质细胞瘤肿瘤生长。

结论：据我们所知，这是第一项提供 CLEC19A 作为 GBM 中潜在肿瘤抑制基因的计算机、分子、细胞和体内证据的报告。这些结果增强了我们对 CLEC19A 在神经胶质瘤中的作用的理解，并证明了 CLEC19A 作为 GBM 潜在治疗靶点的进一步探索。