Orai1对鼻咽癌细胞侵袭和上皮-间充质转化的影响及其机制

目的： 探讨Orai1对鼻咽癌细胞侵袭和上皮-间充质转化的影响及其作用机制。方法： 培养鼻咽上皮细胞NP69和4种鼻咽癌细胞系（CNE1、CNE2、HONE1和5-8F），分别采用实时荧光定量PCR（qPCR）和Western blot法检测细胞中Orai1的mRNA和蛋白表达水平。采用靶向Orai1的短发夹RNA（shRNA）质粒，转染CNE2细胞，设转染对照质粒的CNE2细胞为阴性对照组，钙离子成像检测钙库调节的钙离子内流，Transwell小室检测细胞转移和侵袭，qPCR和Western blot法检测上皮-间充质转化（EMT）标志物E钙黏蛋白（E-cadherin）、N钙黏蛋白（N-cadherin）和波形蛋白（Vimentin）的mRNA和蛋白表达水平。结果： NP69、CNE1、CNE2、HONE1和5-8F细胞中均表达Orai1，且CNE2细胞中表达相对较高，故选择CNE2细胞进行后续试验。与阴性对照组相比，转染Orai1 shRNA后，CNE2细胞中Orai1的mRNA和蛋白水平均显著降低（P<0.05）；CNE2细胞的钙库调节钙离子内流能力、转移/侵袭能力和EMT相关标志蛋白E-cadherin、N-cadherin和Vimentin的mRNA和蛋白表达水平均显著下降（P<0.05）。结论： 抑制Orai1表达可抑制鼻咽癌细胞钙库调节钙离子内流、转移侵袭和EMT进程，提示Orai1可能成为治疗鼻咽癌转移的新靶标和新方向。

Mechanisms of Orai1 on invasion and epithelial-mesenchymal transformation in nasopharyngeal carcinoma cells

OBJECTIVE: The study was aimed to investigate mechanisms of Orai1 on nasopharyngeal carcinoma cell invasion and epithelial-mesenchymal transformation (EMT) in vitro. METHODS: A nasopharyngeal epithelial cell line (NP69) and nasopharyngeal carcinoma cell lines (CNE1,CNE2,HONE1 and 5-8F) were cultured. Real-time quantitative PCR and Western blot were used to detect expressions of Orai1 mRNA and protein. The short hairpin RNA (shRNA) plasmid targeting Orai1 was used to down-regulate expressions of Orai1 in CNE2 cells. Store-operated Ca²⁺ entry (SOCE) was detected by calcium imaging,cell migration and invasion were detected by Transwell chamber,and E cadherin,N cadherin and Vimentin were detected by real-time quantitative PCR and Western blot. RESULTS: Orai1 was expressed in NP69,CNE1,CNE2,HONE1 and 5-8F cells,and the expression of Orai1 was higher in CNE2 cells than that in NP69 cells (P<0.05). The mRNA and protein levels of ORAI1 were significantly decreased in CNE2 cells transfected with Orai1 shRNA (P<0.05). SOCE,migration/invasion and EMT were significantly inhibited in Orai1 shRNA transfected CNE2 cells (P<0.05). CONCLUSION: Blockage of ORAI1 expression inhibited SOCE,migration/invasion and EMT process of nasopharyngeal carcinoma cells. Therefore, Orai1 might be a new target and a new direction for the treatment of nasopharyngeal carcinoma metastasis.