红景天苷通过miR-99a-5p/IGF-1R信号通路调节鼻咽癌细胞的增殖、凋亡、迁移和侵袭

目的:探讨红景天苷调节微小RNA-99a-5p（microRNA-99a-5p，miR-99a-5p）/胰岛素样生长因子1受体（insulin-like growth factor 1 receptor，IGF-1R）信号通路对鼻咽癌细胞增殖、凋亡、迁移和侵袭的影响。方法:实时荧光定量PCR（qRT-PCR）法检测人鼻咽上皮细胞（NP69）、鼻咽癌细胞系（CNE-2Z、HONE1、6-10B、HNE2）中miR-99a-5p、IGF-1R信使RNA（mRNA）表达水平；将对数期HNE2细胞分为对照组（常规培养HNE2细胞）、红景天苷低浓度组（含HNE2细胞的培养基中加入0.5 μg/mL的红景天苷）、红景天苷中浓度组（含HNE2细胞的培养基中加入1 μg/mL的红景天苷）、红景天苷高浓度组（含HNE2细胞的培养基中加入2 μg/mL的红景天苷）、模拟物（mimics）-NC组（HNE2细胞转染miR-99a-5p mimics阴性对照）、miR-99a-5p mimics组（HNE2细胞转染miR-99a-5p mimics）、红景天苷高浓度+抑制剂（inhibitor）NC组（含HNE2细胞的培养基中加入2 μg/mL的红景天苷并转染miR-99a-5p inhibitor阴性对照）、红景天苷高浓度+miR-99a-5p inhibitor组（含HNE2细胞的培养基中加入2 μg/mL的红景天苷并转染miR-99a-5p inhibitor）。qRT-PCR法检测各组HNE2细胞中miR-99a-5p、IGF-1R mRNA表达水平；细胞计数试剂盒-8、流式细胞术、Transwell小室、划痕实验、蛋白免疫印迹法分别检测HNE2细胞增殖能力、凋亡率、侵袭能力、迁移能力、IGF-1R蛋白表达水平；双荧光素酶报告基因实验验证miR-99a-5p与IGF-1R的靶向关系。结果:与NP69细胞相比，CNE-2Z、HONE1、6-10B、HNE2细胞系中miR-99a-5p表达水平降低，IGF-1R mRNA表达水平升高（P＜0.05），其中HNE2细胞中miR-99a-5p、IGF-1R mRNA表达水平与NP69细胞差异更明显（P＜0.05）；与对照组相比，红景天苷低、中、高浓度组miR-99a-5p表达水平、增殖抑制率、凋亡率依次升高（P＜0.05），IGF-1R mRNA与蛋白表达水平、侵袭细胞数目、划痕愈合率依次降低（P＜0.05）；与对照组、mimics-NC组相比，miR-99a-5p mimics组miR-99a-5p表达水平、增殖抑制率、凋亡率升高（P＜0.05），IGF-1R mRNA与蛋白表达水平、侵袭细胞数目、划痕愈合率降低（P＜0.05）；与红景天苷高浓度组、红景天苷高浓度+inhibitor NC组相比，红景天苷高浓度+miR-99a-5p inhibitor组miR-99a-5p表达水平、增殖抑制率、凋亡率降低（P＜0.05），IGF-1R mRNA与蛋白表达水平、侵袭细胞数目、划痕愈合率升高（P＜0.05）；miR-99a-5p与IGF-1R存在靶向关系。结论:红景天苷可能通过促进miR-99a-5p表达，靶向抑制IGF-1R表达，参与抑制HNE2细胞增殖、侵袭、迁移，并促进HNE2细胞凋亡。

Salidroside regulates proliferation,apoptosis,migration and invasion of nasopharyngeal carcinoma cells via miR-99a-5p/IGF-1R signaling pathway

Objective:To investigate the effects of salidroside on the proliferation,apoptosis,migration and invasion of nasopharyngeal carcinoma cells by regulating microRNA-99a-5p (miR-99a-5p)/insulin-like growth factor 1 receptor (IGF-1R) signaling pathway.Methods:Real time fluorescence quantitative PCR (qRT-PCR) was used to detect the expression levels of miR-99a-5p and IGF-1R messenger RNA (mRNA) in human nasopharyngeal epithelial cells (NP69),nasopharyngeal carcinoma cell lines (CNE-2Z,HONE1,6-10B,HNE2).The logarithmic phase HNE2 cells were divided into control group (conventional culture of HNE2 cells),salidroside low concentration group (0.5 μg/mL salidroside was added to the medium containing HNE2 cells),salidroside medium concentration group (1 μg/mL salidroside was added to the medium containing HNE2 cells),salidroside high concentration group (2 μg/mL salidroside was added to the medium containing HNE2 cells),mimics-NC group (HNE2 cells were transfected with miR-99a-5p mimics negative control),miR-99a-5p mimics group (HNE2 cells were transfected with miR-99a-5p mimics),salidroside high concentration+inhibitor NC group (2 μg/mL salidroside was added to the medium containing HNE2 cells and transfected with miR-99a-5p inhibitor negative control),salidroside high concentration+miR-99a-5p inhibitor group (2 μg/mL salidroside was added to the medium containing HNE2 cells and transfected with miR-99a-5p inhibitor).The expression levels of miR-99a-5p and IGF-1R mRNA in HNE2 cells of each group were detected by qRT-PCR.Cell counting kit-8,flow cytometry,Transwell chamber,scratch test and Western blot were used to detect the proliferation ability,apoptosis rate,invasion ability,migration ability and IGF-1R protein expression levels of HNE2 cells.Dual-luciferase reporter gene experiment was used to verify the targeting relationship between miR-99a-5p and IGF-1R.Results:Compared with NP69 cells,the expression level of miR-99a-5p decreased and the expression level of IGF-1R mRNA increased in CNE-2Z,HONE1,6-10B and HNE2 cell lines (P＜0.05).The expression levels of miR-99a-5p and IGF-1R mRNA in HNE2 cells were significantly different from those in NP69 cells (P＜0.05).Compared with the control group,the expression level of miR-99a-5p,proliferation inhibition rate and apoptosis rate in salidroside low,medium and high concentration groups were increased in turn (P＜0.05),and the expression levels of IGF-1R mRNA and protein,the number of invasive cells and scratch healing rate were decreased in turn (P＜0.05).Compared with the control group and mimics-NC group,the expression level of miR-99a-5p,proliferation inhibition rate and apoptosis rate in miR-99a-5p mimics group were increased (P＜0.05),and the expression levels of IGF-1R mRNA and protein,the number of invasive cells and scratch healing rate were decreased (P＜0.05).Compared with salidroside high concentration group and salidroside high concentration+inhibitor NC group,the expression level of miR-99a-5p,proliferation inhibition rate and apoptosis rate in salidroside high concentration+miR-99a-5p inhibitor group were decreased (P＜0.05),and the expression levels of IGF-1R mRNA and protein,the number of invasive cells and scratch healing rate were increased (P＜0.05).miR-99a-5p had a targeting relationship with IGF-1R.Conclusion:Salidroside may promote the expression of miR-99a-5p and target and inhibit IGF-1R expression to inhibit the proliferation,invasion and migration of HNE2 cells,and promote the apoptosis of HNE2 cells.