摘要
目的探究N6-甲基腺苷(N6-methyladenosine,m^(6)A)修饰调控RP11-426A6.5在喉鳞状细胞癌(laryngeal squamous cell carcinoma,LSCC)发生与发展过程中的作用机制。方法收集2017年1月至9月哈尔滨医科大学附属第二医院耳鼻咽喉头颈外科收治的48例LSCC患者的癌及癌旁组织,选取其中3例LSCC样本利用长链非编码RNA(long non-coding RNA,lncRNA)m^(6)A甲基化芯片及表达谱芯片技术检测lncRNAs的甲基化和表达水平并筛选关键lncRNA。通过甲基化RNA免疫共沉淀-定量PCR(methylated RNA immunoprecipitation-quantitative PCR,MeRIP-qPCR)及实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)在其余45例LSCC样本中验证RP11-426A6.5的m^(6)A修饰和表达水平,并分析RP11-426A6.5与肿瘤恶性程度、预后等临床因素间的相关性。在体外,运用RNA干扰(RNA interference,RNAi)技术构建低表达RP11-426A6.5喉癌细胞系,以EdU细胞增殖检测实验、伤口愈合实验、Transwell侵袭实验分别观察LSCC细胞的增殖、迁移、侵袭等生物学功能变化,以裸鼠成瘤实验验证RP11-426A6.5下调后对体内移植瘤生长的影响。应用TCGA数据库及SRAMP网站预测介导RP11-426A6.5 m^(6)A修饰的相关酶及相应甲基化位点,选用MazF酶切法对结合位点验证。运用RNAi技术在细胞水平干扰修饰酶相应基因表达后观察细胞功能的改变并利用MeRIP-qPCR检测RP11-426A6.5 m^(6)A水平变化,以放射线菌素D处理细胞系观察RP11-426A6.5稳定性改变。结果LSCC组织中RP11-426A6.5甲基化及表达水平明显高于癌旁组织[RP11-426A6.5甲基化水平:癌组织(23.828±4.975)比癌旁组织(20.280±3.607),RP11-426A6.5表达水平:癌组织(1.197±0.314)比癌旁组织(1.015±0.170),P值均<0.05],且RP11-426A6.5表达水平与肿瘤大小(T期)密切相关[T1-2期(1.081±0.298)比T3-4期(1.306±0.292),χ2=5.35,P<0.05]。RP11-426A6.5高表达患者的术后生存期明显低于低表达组患者,差异有统计学意义(P=0.046)。体内外试验结�
Objective To investigate the roles of N6-methyladenosine(m^(6)A)modification in regulating RP11-426A6.5 in the development of laryngeal squamous cell carcinoma(LSCC).Methods The methylation and expression levels of lncRNAs were identified and important lncRNAs were screened utilizing long non-coding RNA(lncRNA)m^(6)A methylation microarray.Cancer and para cancer tissue samples were taken from 48 LSCC patients hospitalized to the Department of Otolaryngology-Head and Neck Surgery of the Second Affiliated Hospital of Harbin Medical University between January and September 2017.Expression profiling microarray was performed in 3 of 48 LSCC samples,and methylated RNA immunoprecipitation-quantitative PCR(MeRIP-qPCR)and quantitative real-time fluorescent PCR(qRT-PCR)were performed in the remaining 45 LSCC samples to verify the m^(6)A modification and expression levels of RP11-426A6.5.Correlations between RP11-426A6.5 and clinical factors were anlysed.Laryngeal cancer cell line with low expression of RP11-426A6.5 was created in vitro using RNA interference(RNAi)technology.The 5-Ethynyl-2′-deoxyuridine(EdU)cell proliferation experiment,wound healing experiment,and transwell invasion experiment were used respectively to measure the proliferation,migration,and invasion of LSCC cells.The effect of RP11-426A6.5 down-regulation on the growth of transplanted tumors in vivo was verified by nude mice tumorigenesis assay.The Cancer Genome Atlas(TCGA)database and sequence-based RNA adenosine methylation site predictor(SRAMP)website were used to predict the enzymes and corresponding methylation sites.MazF digestion was chosen to validate the binding sites.RNAi technology was used to observe the changes in cell function after interfering with the expression of the corresponding genes of the modified enzymes.MeRIP-qPCR was used to detect the level of RP11-426A6.5 m^(6)A cell line treated with actinomycin D was used to observe the stability of RP11-426A6.5.Results RP11-426A6.5 methylation and expression levels were significantly high
作者
阎柄睿
王鹏
李雨珊
杨丽可
李秋影
阚轩
王婧婷
孙亚男
Yan Bingrui;Wang Peng;Li Yushan;Yang Like;Li Qiuying;Kan Xuan;Wang Jingting;Sun Yanan(The Second Affiliated Hospital of Harbin Medical University,Department of Otolaryngology-Head and Neck Surgery,Harbin 150001,China)
出处
《中华耳鼻咽喉头颈外科杂志》
CSCD
北大核心
2022年第12期1470-1478,共9页
Chinese Journal of Otorhinolaryngology Head and Neck Surgery
