Supplementary Material for: ABCF1/CXCL12/CXCR4 enhances glioblastoma cell proliferation, migration and invasion by activating the PI3K/AKT signal pathway

Background: Glioblastoma (GBM) is the most prevalent and fatal form of brain tumor, which is associated with a poor prognosis. ATP-binding cassette subfamily F member 1 (ABCF1), is an E2 ubiquitin-conjugating enzyme, which is implicated in regulating immune responses and tumorigenesis. Aberrant E3 ubiquitylation has been evidenced in GBM. However, the role of ABCF1 in GBM needs to be further explored. Methods: The expression of ABCF1, CXC chemokine ligand 12 (CXCL12) and CXC chemokine receptor 4 (CXCR4) in GBM tissues was examined by the GEPIA tool, real-time PCR and western blotting. HMC3, U251MG and LN-229 cells were cultured and transfected with shRNA targeting ABCF1 and ABCF1 plasmid. The proliferative, migrative and invasive ability of cells was detected. Western blotting was used to detect the levels of phosphorylated phosphatidylinositol 3-kinase (PI3K) and phosphorylated protein kinase B (AKT). Results: We observed that GBM tissues had higher ABCF1, CXCL12, and CXCR4 expression levels. The expression of CXCL12 and CXCR4were enhanced by ABCF1 overexpression, which were significantly reversed by silence of ABCF1 in GBM cells. Silencing ABCF1 or CXCR4 inhibition weakened the capacity of GBM cell growth, migration and invasion, while ectopic ABCF1 expression or CXCL12 treatment enhanced the cellular function of GBM cells. Furthermore, p-PI3K and p-AKT protein levels were downregulated by ABCF1 knockdown or CXCR4 blockade, which were prompted by ABCF1 overexpression or CXCL12 supplement. Conclusion: The ABCF1-CXCL12-CXCR4 axis was identified as a key player in GBM cell survival and metastasis through activating the PI3K/AKT signaling pathway in GBM cells.

背景：胶质母细胞瘤（Glioblastoma, GBM）是最常见且致死性最高的脑部肿瘤类型，预后极差。ATP结合盒亚家族F成员1（ATP-binding cassette subfamily F member 1, ABCF1）属于E2泛素结合酶，参与调控免疫应答与肿瘤发生过程。已有研究证实，异常E3泛素化与胶质母细胞瘤的发生发展密切相关，但ABCF1在胶质母细胞瘤中的具体作用仍有待进一步探究。
方法：本研究通过GEPIA工具、实时荧光定量PCR（real-time PCR）与蛋白质印迹法（western blotting）检测了胶质母细胞瘤组织中ABCF1、CXC趋化因子配体12（CXC chemokine ligand 12, CXCL12）以及CXC趋化因子受体4（CXC chemokine receptor 4, CXCR4）的表达水平。培养HMC3、U251MG及LN-229细胞，分别转染靶向ABCF1的短发夹RNA（shRNA）与ABCF1过表达质粒。通过相关实验检测细胞的增殖、迁移与侵袭能力；采用蛋白质印迹法检测磷酸化磷脂酰肌醇3-激酶（phosphorylated phosphatidylinositol 3-kinase, PI3K）与磷酸化蛋白激酶B（phosphorylated protein kinase B, AKT）的蛋白表达水平。
结果：本研究观察到，胶质母细胞瘤组织中ABCF1、CXCL12及CXCR4的表达水平显著升高。ABCF1过表达可上调CXCL12与CXCR4的表达，而沉默ABCF1则可显著逆转该效应。在胶质母细胞瘤细胞中，沉默ABCF1或抑制CXCR4均可削弱细胞的增殖、迁移与侵袭能力；反之，过表达ABCF1或施加CXCL12处理则可增强胶质母细胞瘤细胞的上述细胞功能。此外，ABCF1敲低或CXCR4阻断可下调p-PI3K与p-AKT的蛋白表达水平，而ABCF1过表达或添加CXCL12则可上调二者的表达水平。
结论：本研究证实，ABCF1-CXCL12-CXCR4轴通过激活胶质母细胞瘤细胞内的PI3K/AKT信号通路，在肿瘤细胞存活与转移过程中发挥关键调控作用。