ABSTRACT

Store-operated Ca²⁺ entry (SOCE) plays an important role in regulating Ca²⁺ influx, which participates in tumor cell survival and motility. We aim to elucidate the role of SOCE in the behavior of C6 glioma cells. Lentiviral vector inserted with the Orai1-targeting shRNA was used to inhibit SOCE in C6 glioma cells. The down-regulation of Orai1 was confirmed by western blot. The ability of shOrai1 or SOCE inhibitor (SKF96365) in regulating SOCE inhibition was evaluated by measuring Ca²⁺ concentration. Additionally, its effect on cell behavior was assessed using methyl thiazolyl tetrazolium (MTT) assay, wound healing assay, transwell assay, and adhesion assay. Focal adhesions were visualized by immunofluorescence assay. Further, the expression of proline-rich tyrosine kinase 2 (Pyk2) and phosphorylated Pyk2 (p-Pyk2) was analyzed using western blot. Both, SKF96365 treatment and the Orai1 down-regulation inhibited SOCE by perturbing Ca²⁺ influx. The inhibitory effects of shOrai1 on C6 cell proliferation, migration, and invasion were similar to that of SKF96365. Moreover, Orai1 inhibition enhanced C6 cell adhesion by increasing the size of focal adhesion plaques. The down-regulation of Pyk2 was observed in both SKF96365-treated and Orai1-silenced C6 cells. Additionally, Orai1 inhibition blocked AKT/mTOR, NFAT, and NF-κB pathways. The silencing of Orai1 inhibited the C6 glioma cell migration, invasion and contributed to focal adhesion.

摘要

Store-operated Ca ²⁺ entry (SOCE) 在调节 Ca ²⁺流入方面发挥重要作用，Ca ²⁺流入参与肿瘤细胞存活和运动。我们旨在阐明 SOCE 在 C6 神经胶质瘤细胞行为中的作用。插入了靶向 Orai1 的 shRNA 的慢病毒载体用于抑制 C6 神经胶质瘤细胞中的 SOCE。通过蛋白质印迹证实了 Orai1 的下调。通过测量Ca ²⁺评估shOrai1或SOCE抑制剂（SKF96365）调节SOCE抑制的能力专注。此外，使用甲基噻唑基四唑 (MTT) 测定、伤口愈合测定、transwell 测定和粘附测定评估了其对细胞行为的影响。通过免疫荧光测定可视化粘着斑。此外，使用蛋白质印迹分析富含脯氨酸的酪氨酸激酶 2 (Pyk2) 和磷酸化 Pyk2 (p-Pyk2) 的表达。SKF96365 处理和 Orai1 下调都通过扰乱 Ca ^{2+ 来}抑制 SOCE涌入。shOrai1对C6细胞增殖、迁移和侵袭的抑制作用与SKF96365相似。此外，Orai1 抑制通过增加粘着斑的大小来增强 C6 细胞粘附。在 SKF96365 处理和 Orai1 沉默的 C6 细胞中均观察到 Pyk2 的下调。此外，Orai1 抑制阻断了 AKT/mTOR、NFAT 和 NF-κB 通路。Orai1 的沉默抑制了 C6 神经胶质瘤细胞的迁移、侵袭并有助于粘着斑。