lncRNA NEAT1降表达人喉鳞状细胞癌细胞增殖凋亡变化及与miR-214靶向关系  

Changes in proliferation and apoptosis of human laryngeal squamous cell carcinoma cells with down-expression of LncRNA NEAT1 and its targeted relationship with miR-214

作　　者：罗德艳[1,2] 冯俊 彭涛[1,2] 唐一萍 杨久梅[1,2] LUO Deyan;FENG Jun;PENG Tao;TANG Yiping;YANG Jiumei(The Second Clinical Medical College of North Sichuan Medical College,Nanchong 637000,China;不详)

机构地区：[1]川北医学院第二临床医学院,四川南充637000 [2]南充市中心医院耳鼻咽喉头颈外科

出　　处：《山东医药》2024年第18期26-30,共5页Shandong Medical Journal

基　　金：四川省科技厅重点研发项目(2018FZ0116);四川省教育厅自然科学重点项目(18ZA203);南充市科技局应用技术研究与开发基金项目(16YFZJ0021)。

摘　　要：目的观察长链非编码RNA(lncRNA)核富集转录体1(NEAT1)降表达的人喉鳞状细胞癌(LSCC)细胞增殖凋亡变化,探讨其与微小RNA-214(miR-214)的靶向关系。方法采用实时荧光定量PCR(RT-qPCR)法检测人永生化表皮细胞Hacat和人LSCC细胞系(FD-LSC-1、Hep-2、TU177)中lncRNA NEAT1、miR-214,选择TU177细胞为受试细胞。取对数生长期TU177细胞,分为甲、乙组,分别转染si-lncRNA NEAT1(敲低lncRNA NEAT1表达)、si-NC(乱序无意义序列),培养至24 h时采用CCK8法观察两组细胞增殖情况、采用平板克隆形成实验观察两组细胞克隆形成情况、采用流式细胞术观察两组细胞周期和细胞凋亡情况、采用RT-qPCR法检测两组细胞lncRNA NEAT1及miR-214。另取对数生长期TU177细胞,分为一二三四组,一组顺序转染WT-lncRNA NEAT1、miR-214 mimic,二组顺序转染WT-lncRNA NEAT1、miR-NC,三组顺序转染MUT-lncRNA NEAT1、miR-214 mimics,四组顺序转染MUT-lncRNA NEAT1、miR-NC。采用双荧光素酶报告基因实验验证lncRNA NEAT1及miR-214的靶向关系。结果与乙组相比,培养24、48、72 h时甲组TU177细胞OD值低,培养14 d时甲组TU177细胞克隆形成数少,甲组TU177细胞G0/G1期细胞比例高、S期细胞比例低,细胞凋亡率高(P均<0.05);与乙组相比,转染24 h时甲组TU177细胞lncRNA NEAT1相对表达量低、miR-214相对表达量高(P均<0.05)。培养48 h时一、二、三、四组TU177细胞细胞荧光素酶活性分别为0.63±0.08、0.99±0.01、1.02±0.02、0.98±0.03,与二组相比,一组细胞荧光素酶活性低(P<0.05)。结论敲低lncRNA NEAT1表达可抑制TU177细胞的增殖和克隆,阻滞细胞周期于G0/G1期,并促进细胞的凋亡。TU177细胞中lncRNA NEAT1与miR-214靶向相关。lncRNA NEAT1可能通过与miR-214靶向结合,抑制TU177细胞的增殖克隆,阻滞细胞周期于G0/G1期,促进细胞的凋亡。Objective To observe the effects of long non-coding RNA(lncRNA)nuclear-enriched abundant tran⁃script 1(NEAT1)on the proliferation and apoptosis of human laryngeal squamous cell carcinoma(LSCC)cells,as well as its targeted relationship with microRNA-214(miR-214).Methods Real-time fluorescence quantitative PCR(RT-qP⁃CR)was used to detect lncRNA NEAT1 and miR-214 in immortalized human epidermal cells Hacat and human LSCC cell lines(FD-LSC-1,Hep-2,and TU177),and then TU177 cells were selected as the experimental cells.TU177 cells in the logarithmic growth phase were divided into groups A and B,which were transfected with si-lncRNA NEAT1(knockdown of lncRNA NEAT1 expression)and si-NC(random meaningless sequence),respectively.At 24 h,cell proliferation was ob⁃served using CCK-8 assay,colony formation experiment was conducted to observe clone formation,flow cytometry was used to analyze cell cycle distribution and apoptosis rate,while RT-qPCR was performed to detect lncRNA NEAT1 and miR-214 expression in both groups.TU177 cells in the logarithmic growth phase were divided into groupsⅠ,Ⅱ,ⅢandⅣ,which were transfected with WT-lncRNA NEAT1 and miR-214 mimic,WT-lncRNA NEAT1 and miR-NC,MUT-lncRNA NEAT1 and miR-214 mimic,and MUT-lncRNA NEAT1 and miR-NC,respectively.Th targeted relationship between ln⁃cRNA NEAT1 and miR-214 was validated by dual luciferase reporter gene assay.Results Compared with the group B,the OD values of TU177 cells were lower at 24,48,and 72 h of cultivation;at 14 d of cultivation,the number of clone for⁃mation in TU177 cells was smaller;the proportion of cells in the G0/G1 phase was higher,the proportion of cells in the S phase was lower,and the apoptosis rate was higher in the group A(all P<0.05).Compared with the group B,at 24 h of trans⁃fection,the relative expression of lncRNA NEAT1 was lower and the relative expression of miR-214 in TU177 cells was higher in the group A(both P<0.05).At 48 h of cultivation,the luciferase activity of TU177 cells in the groupsⅠ,Ⅱ,ⅢandⅣwas 0.

关 键 词：长链非编码RNA 长链非编码RNA核富集转录体1 微小RNA-214 喉鳞状细胞癌 细胞增殖 细胞凋亡 细胞克隆 细胞周期 

分 类 号：R739[医药卫生—肿瘤]