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. 2020 Dec;19(24):3468-3479.
doi: 10.1080/15384101.2020.1843814. Epub 2020 Dec 3.

Suppression of store-operated Ca2+ entry regulated by silencing Orai1 inhibits C6 glioma cell motility via decreasing Pyk2 activity and promoting focal adhesion

Meng Zhu  1   2 , Bingke Lv  1 , Wenjing Ge  3 , Zhenwen Cui  1 , Kai Zhao  1 , Yugong Feng  1 , Xuejun Yang  2
Affiliations

Suppression of store-operated Ca2+ entry regulated by silencing Orai1 inhibits C6 glioma cell motility via decreasing Pyk2 activity and promoting focal adhesion

Meng Zhu et al. Cell Cycle. 2020 Dec.

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.

Keywords: Store-operated Ca2+ entry; calcium release-activated calcium channel protein1; cell adhesion; glioma; proline-rich tyrosine kinase 2.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1.
Figure 1.
Effect of Orai1 suppression on SOCE in C6 glioma cells. (a) The glycosylated and non-glycosylated protein expression of Orai1 was measured in C6 glioma cells transfected with shControl, shScramble or shOrai1 by western blot analysis. Data are shown as means ± SDs from thrice independent experiments. ** P< 0.01 vs. the shControl group. (b) C6 glioma cells were treated with 0–30 μM of SKF96365 and the proliferation ratio was detected by MTT assay. (c) The Ca2+ influx curves, prepared with 25–45 single cells transfected with shOrai1/shScramble or treated with SKF96365 (20 µM) by using Fluo-4/AM-based Ca2+ measurements. TG, thapsigargin
Figure 2.
Figure 2.
Effect of Orai1 suppression on C6 glioma cell proliferation. C6 glioma cells were transfected with shScramble/shOrai1 or treated with SKF96365 (20 µM). (a) The relative proliferation ratio of C6 cells was determined by MTT assay. (b) Cell cycle distribution was determined by flow cytometry. (c) Cells were analyzed for expression of p21, Cyclin D1, and CDK4 protein with Western blot. Data are shown as means ± SDs from thrice independent experiments. *P < 0.05 or ** P< 0.01 vs. the shScramble group
Figure 3.
Figure 3.
Effect of Orai1 suppression on C6 glioma cell migration and invasion. C6 glioma cells were transfected with shScramble/shOrai1 or treated with SKF96365 (20 µM). (a) The migratory areas of three groups of C6 cells were marked and measured by utilizing the wound healing assay (magnification, ×40). (b) The invasive ability of C6 cells was determined by utilizing Matrigel-coated transwell assay (magnification, ×100). (c) Cells were analyzed for expression of E-cadherin, N-cadherin and Vimentin protein with Western blot. Data are shown as means ± SDs from thrice independent experiments. * P < 0.05 or ** P < 0.01 vs. the shScramble group
Figure 4.
Figure 4.
Effect of Orai1 suppression on C6 glioma cell adhesion and Pyk2 phosphorylation. shScramble or shOrai1 was introduced into C6 glioma cells, or cells were treated with SKF96365 (20 µM). (a) The relative cell adhesion was detected. (b) Cell morphology was visualized by immunofluorescence assay and the proportions of cells with large focal adhesions were calculated.(c) The protein levels of total Pyk2 and phosphorylated Pyk2 (p-Pyk2) were measured by western blot analysis. Data are shown as means ± SDs from thrice independent experiments. * P < 0.05 or ** P < 0.01 vs. the shScramble group
Figure 5.
Figure 5.
Effect of Orai1 down-regulation on signal pathways in C6 glioma cells. C6 glioma cells were transfected with shScramble/shOrai1 or treated with SKF96365 (20 µM). Cells were analyzed for expression levels of AKT phosphorylated protein, 4EBP1 phosphorylated protein, NFAT protein, NF-κB protein and S6K phosphorylated protein with Western blot
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This work was supported by the National Natural Science Foundation of China [No. 81872063 and No. 81472352 and No.81901249].