miR-454-3p对鼻咽癌CNE-2细胞上皮-间质转化的调控机制

Effect of miR-454-3p on regulating epithelial-mesenchymal transition of nasopharyngeal carcinoma cells and its mechanism

目的探讨miR-454-3p对鼻咽癌CNE-2细胞上皮-间质转化(EMT)的调控机制。方法收集2017年1月至2018年12月柳州市中医医院就诊的95例鼻咽癌患者的血清和鼻咽癌组织标本及健康体检者30例的血清,采用RT-qPCR检测miR-454-3p和E-盒结合锌指蛋白2(ZEB2)血清水平和mRNA的表达,对鼻咽癌组织中miR-454-3p和ZEB2 mRNA的表达进行相关性分析。将CNE-2细胞随机分为Control组、miR-454-3p mimic组、Mimic-NC组、miR-454-3p mimic+ZEB2 plasmid组和miR-454-3p mimic+vector组,采用Transwell实验检测细胞侵袭能力,细胞划痕实验检测细胞迁移能力,Western blot检测ZEB2、E-cadherin、N-cadherin、Vimentin蛋白表达。双荧光素酶实验验证miR-454-3p与ZEB2的靶向关系。结果鼻咽癌患者血清miR-454-3p水平低于健康体检者,差异有统计学意义(P<0.05)。鼻咽癌Ⅲ、Ⅳ期患者鼻咽癌组织miR-454-3p相对表达水平低于Ⅰ、Ⅱ期患者,ZEB2 mRNA相对表达水平则升高,差异均有统计学意义(均P<0.05)。miR-454-3p与ZEB2 mRNA的表达呈负相关(r=-0.734,P<0.01);与Mimic-NC组比较,miR-454-3p mimic组ZEB2、N-cadherin、Vimentin表达水平均降低,E-cadherin表达水平升高,侵袭细胞数及迁移率均降低,差异均有统计学意义(均P<0.05),过表达ZEB2可逆转miR-454-3p mimie对EMT的抑制作用。双荧光素酶实验显示miR-454-3p可靶向ZEB23'UTR。结论miR-454-3p在鼻咽癌患者血清及癌组织中呈低表达,可通过靶向调控ZEB2的表达水平进而抑制鼻咽癌细胞EMT。

Objective To investigate the effect of miR-454-3p on epithelial-mesenchymal transition(EMT)of nasophary-ngeal carcinoma(NPC)cells and its regulatory mechanism.Methods The serum and tumor tissues of 95 NPC patients treated in our hospital from January 2017 to December 2018 were collected,and 30 serum samples from healthy subjects were used as controls.qRT-PCR was applied to detect the expression of miR-454-3p and E-box-bound zinc finger protein 2(ZEB2)in NPC tissues,the correlation of miR-454-3p and ZEB2 expressions was analyzed with Spearman's correlation analysis.The NPC CNE-2 cells were divided into the Control,miR-454-3p mimic,Mimic-NC,miR-454-3p mimic+ZEB2plasmid and miR-454-3p mimic+vector group.The invasive ability of cells was detected by Transwell assay,the ability of cell migration was detected by cell scratch method.Western blot was used to detect the proteins expression of ZEB2,E-cadherin,N-cadherin and Vimentin.The targeting relationship between miR-454-3p and ZEB2 was verified by double luciferase assay.Results Expression of miR-454-3p in serum of NPC patients was lower than that of controls(P<0.05).The expression of miR-454-3p in patients with stageⅢ&ⅣNPC was lower than that in patients with stageⅠ&ⅡNPC,while the expression of ZEB2 mRNA was increased,The expression of miR-454-3p was negatively correlated with ZEB2 mRNA(P<0.05).Compared with Control and Mimic-NC group,the expression of ZEB2,N-cadherin and Vimentin in the miR-454-3p mimic group were reduced,the expression of E-cadherin was increased,and the number of invasive cells and the rate of cell migration were reduced,overexpression of ZEB2 reversed the inhibitory effect of miR-454-3p mimic on EMT(all P<0.05).Double luciferase assay showed that miR-454-3p could target ZEB23'UTR.Conclusion MiR-454-3p expression is down-regulated in the serum and cancer tissues of patients with nasopharyngeal carcinoma,and it can inhibit the EMT of NPC CNE-2 cells by targeting the expression of ZEB2.