LncRNA HOTAIR通过调控miR-30d影响鼻咽癌细胞的侵袭和迁移

Influence of LncRNA HOTAIR on invasion and migration of nasopharyngeal carcinoma cells by regulating miR-30d

目的探讨长链非编码RNA(LncRNA)HOX转录本反义RNA(HOTAIR)通过调控靶向小分子RNA-30d(miR-30d)影响鼻咽癌细胞侵袭和迁移的机制。方法在鼻咽癌CNE-2细胞中转染HOTAIRsiRNA,使用实时定量聚合酶链反应(RT-qPCR)测定转染效果,Transwell法测定肿瘤细胞侵袭及迁移能力,蛋白质免疫印迹(Western blot)检测细胞中上皮型钙黏蛋白(E-cadherin)、波形蛋白(vimentin)和葡萄糖调节蛋白78(GRP78)的表达;生物信息学软件分析HOTAIR与miR-30d区域结合位点,利用双荧光素酶报告系统确定两者结合关系;用RT-qPCR方法检测下调HOTAIR后鼻咽癌细胞中miR-30d表达的变化。将HOTAIR siRNA和miR-30d inhibitor共转染至CNE-2细胞中,用上述方法分析细胞侵袭、迁移及E-cadherin、vimentin和GRP78蛋白表达的变化。结果敲减HOTAIR表达后,CNE-2细胞的侵袭和迁移能力下降,细胞中vimentin和GRP78蛋白水平降低,E-cadherin蛋白水平升高;HOTAIR靶向调控miR-30d的表达,敲减HOTAIR的表达可以提高CNE-2细胞中miR-30d的水平,miR-30d inhibitor可以明显逆转敲减HOTAIR对CNE-2细胞侵袭、迁移能力以及E-cadherin、vimentin和GRP78蛋白表达的影响。结论HOTAIR通过负调控miR-30d影响GRP78和上皮细胞-间充质转化(EMT)相关蛋白的表达,有利于促进鼻咽癌细胞的侵袭和迁移。

Objective To investigate the mechanism of long-chain non-coding RNA(LncRNA)HOX transcript antisense RNA(HOTAIR)targeting microRNA-30d(miR-30d)on invasion and migration of nasopharyngeal carcinoma cells.Methods HOTAIR siRNA was transfected into CNE-2 cells of nasopharyngeal carcinoma.Realtime quantitative polymerase chain reaction(RT-qPCR)was performed to measure the transfection effect.Transwell assay was carried out to measure the invasion and migration abilities of tumor cell.Western blot was used to detect the expression of epithelial cadherin,vimentin and glucose regulatory protein78(GRP78)in the cells.Bioinformatics software was used to analyze the binding sites of HOTAIR and miR-30 d.The binding relationship was determined by double luciferase reporting system.RT-qPCR was used to detect the change of miR-30d expression after HOTAIR was down-regulated in nasopharyngeal carcinoma cells.HOTAIR siRNA and miR-30d inhibitor were co-transfected into CNE-2 cells.The changes of cell invasion and migration were analyzed by the above methods,as well as the expression of E-cadherin,vimentin and GRP78 proteins.Results After knocking down the expression of HOTAIR,the invasion and migration abilities of CNE-2 cells were decreased;the levels of vimentin and GRP78 were decreased but the level of E-cadherin was increased.HOTAIR regulated specifically the expression of miR-30d.The knock-down of HOTAIR expression could increase the level of miR-30d in CNE-2 cells.The miR-30d inhibitor significantly reversed the effects of HOTAIRsiRNA on invasion and migration abilities of CNE-2 cells and the expression of E-cadherin,vimentin and GRP78 proteins.Conclusion HOTAIR affects the expression of GRP78 and EMT-related proteins by negatively regulating miR-30d,which is beneficial for the invasion and migration of nasopharyngeal carcinoma.