钙通道蛋白Orai1在骨肉瘤细胞转移中的作用研究

目的:骨肉瘤是一种常见的恶性骨肿瘤,恶性程度高,往往在早期就会发生远隔器官的转移,从而导致骨肉瘤的预后非常差。Orai1是一类定位于细胞膜,介导钙离子内流的受体依赖性钙通道蛋白。大量研究发现钙通道蛋白Orai1过表达于多种肿瘤细胞,并对维持肿瘤细胞粘附、侵袭、迁移等恶性表型有非常重要的作用。然而,钙通道蛋白Orai1是否参与了骨肉瘤的转移过程,目前未见相关报道。本研究的目的是探究钙通道蛋白Orai1是否在骨肉瘤转移过程中的发挥作用。方法:利用合成的靶向Orai1的小干扰RNA(Orai1 si RNA)片段,转染至人骨肉瘤细胞系Saos-2细胞。在Saos-2细胞中抑制Orai1的表达。采用细胞黏附实验、细胞划痕实验和细胞Transwell实验检测骨肉瘤细胞的黏附、迁移及侵袭等肿瘤细胞转移能力;Western-blot实验检测Saos-2细胞的中黏着斑激酶(FAK)和桩蛋白(Paxillin)的表达水平和磷酸化水平。结果:靶向Orai1 si RNA瞬时转染至骨肉瘤细胞系Saos-2细胞后,Saos-2细胞中Orai1蛋白表达水平和m RNA转录水平均显著下降。并且,在Saos-2细胞中抑制Orai1表达后,Saos-2细胞的黏附能力、迁移能力、及侵袭能力均显著下降。进一步研究发现,在Saos-2细胞中抑制Orai1表达后,Saos-2细胞的FAK和Paxillin磷酸化水平明显下降。结论:Orai1可以促进骨肉瘤细胞的黏附、迁移和侵袭,增加黏着斑的形成,从而促进骨肉瘤的转移。因此,深入研究钙通道蛋白Orai1调控骨肉瘤转移的分子机制,可为骨肉瘤转移的治疗提供新的新方向和新策略。

The Effect of Orai1 on Metastasis Potential in Osteosarcoma Cells

ABSTRACT Objective: Osteosarcoma is one of the most common skeletal cancers. It is a highly malignant tumor characterized by rapid progression, poor prognosis, and frequent tumor recurrence. Orai1 as calcium channels is widely expressed in various tumor cells, which play essential roles in regulating cell adhesion, migration and invasion. However, it remains unclear whether Orai1is involved in the progression of osteosarcoma. Here, we investigated the functions of Orai1in the metastatic processes of human osteosarcoma cells. Methods: In this study, RNA interference was used to downregulate the expressions of Orai1in human osteosarcoma cells Saos-2. Cell adhere test was used in Orai1siRNA transfected human Saos-2 osteosarcoma cells to test cells adhesion potential. Transwell test was used in Orai1siRNA transfected human Saos-2 osteosarcoma cells to test cells invasion potential. Monolayer wound healing assay was carried out in Orai1siRNA transfected human Saos-2 osteosarcoma cells to test cell migration potential. Western-blot was used to test the phosphorylation levels of FAK and paxillin. Results: Transfection of human osteosarcoma cells with siRNA targeting Orai1, led to significant decreases in Orai1compared to cells transfected with the negative control siRNA. In Orai1siRNA transfected human Saos-2 osteosarcoma cells, the numbers of attached cells, invaded cells and wound closure rate were significantly decreased compared to cells transfected with the negative control siRNA. Furthermore, phosphorylated FAK and Paxillin were markedly lowered in Orai1siRNA transfected human Saos-2 osteosarcoma cells compared to cells transfected with the negative control siRNA. Conclusion: Orai1may play important roles in the invasion and metastasis of human osteosarcoma cells and may promote the formation of focal adhesion. Orai1may enhance metastasis potential of human osteosarcoma cells and may be a promoter of carcinogenesis. Thus, drug targets Orai1may present a new method for preventing osteosarcoma metastasis in osteosarcoma treatments.