喉癌顺铂耐药细胞株RNA测序及整合分析  

RNA sequencing and integration analysis of cisplatin-resistant cell lines of laryngeal carcinoma

作　　者：周毅波[1] 龚小蓉 韩玉杰 刘炜 艾毛毛 张欣睿[1] 于锋[1] ZHOU Yi-bo;GONG Xiao-rong;HAN Yu-jie;LIU Wei;AI Mao-mao;ZHANG Xin-rui;YU Feng(Department of Otolaryngology Head and Neck Surgery,Guangzhou12th People＇s Hospital,Guangzhou Hospital of Otolaryngology Head and Neck Surgery,Institute of Otolaryngology Head and Neck Surgery of Guangzhou Medical University,Guangzhou 510620,P.R.China)

机构地区：[1]广州市第十二人民医院耳鼻咽喉头颈外科.广州市耳鼻咽喉头颈外科医院.广州医科大学耳鼻咽喉头颈外科研究所,广东广州510620

出　　处：《中华肿瘤防治杂志》2018年第13期917-923,共7页Chinese Journal of Cancer Prevention and Treatment

基　　金：广州市科技计划(201604020005;201804010201);广东省医学科研基金(B2018004);广州市医药卫生科技项目(20181A011047)

摘　　要：目的顺铂(cisplatin,DDP)是喉癌患者最常用的化疗药物之一,但是疗效欠佳,可能原因是多药耐药性的产生。本研究将筛选喉癌Hep-2细胞与Hep-2/DDP细胞株之间差异表达的RNA测序数据,包括信使RNA(messenger RNA,mRNA)、长链非编码RNA(long non-coding RNA,lncRNA)及微小RNA(microRNA,miRNA),并整合分析三者之间的相互关系,探讨喉癌多药耐药机制。方法采用HiSeq4000测序平台对Hep-2及Hep-2/DDP细胞的lncRNA+mRNA建库测序,HiSeq2500测序平台进行miRNA建库测序;GenCLiP 2.0分析3种差异表达RNA的功能;并对miRNA与mRNA、lncRNA与miRNA、lncRNA与mRNA分别进行整合分析。RT-PCR实验验证相关miRNA和lncRNA在细胞株之间表达差异。结果 Hep-2及Hep-2/DDP细胞之间差异表达基因共有1 513个转录本,其中上调基因399个,下调基因580个,主要富集的功能有DNA损伤修复、活性氧簇、细胞死亡、凋亡、细胞周期静止和上皮间充质转换等。构建已知的分子相互作用网络的核心节点为上调基因血管内皮生长因子A(vascular endothelial growth factor A,VEGFA)和下调基因蛋白激酶B1(protein kinaseB1,PKB1,又名AKT1);筛选出差异表达的lncRNA共4个,分别是MALAT1、LINC01315、AC092757.3和LINC01822;筛选到63个差异表达miRNA,其中下调miRNA有38个,上调miRNA有25个;整合分析表明,miRNA和lncRNA广泛参与DDP多药耐药机制。RT-PCR结果表明,MALAT1(0.12±0.02)、AC092757.3(0.35±0.02)和MIR155(0.37±0.01)在Hep-2/DDP细胞中表达下调,P值分别为0.036、0.006和0.005;而MIR34A在Hep-2/DDP细胞(3.58±0.01)中表达上调,P=0.011。结论喉癌多药耐药与启动DNA损伤修复、抑制细胞周期以及上皮间充质转化等密切相关,其中VEGFA和AKT1可能是核心基因。miRNA和lncRNA参与DDP多药耐药机制形成,MIR155、MIR34A、MALAT1和AC092757.3可能是主要调节的非编码RNA。OBJECTIVE Cisplatin is one of the most commonly used chemotherapy drugs for patients with laryngeal cancer,but its poor efficacy may be due to the development of multidrug resistance.This study will screen differentially expressed RNA sequencing data between laryngeal cancer Hep-2 cells and Hep-2/DDP cell lines,including messenger RNA（mRNA）and long non-coding RNA（lncRNA）and microRNAs（miRNAs）,and integrate the analysis of the interrelationships among the three to explore the mechanism of multidrug resistance in laryngeal cancer.METHODS The lncRNA ＋ mRNA of Hep-2 and Hep-2/DDP cells were sequenced by HiSeq4000 sequencing platform.The miRNA library was sequenced by HiSeq2500 sequencing platform.GenCLiP 2.0 software was used to analyze the function of three differentially expressed RNAs.mRNA,lncRNA and miRNA,lncRNA and mRNA were integrated analysis.RT-PCR experiments were conducted to verify the differences in the expression of miRNA and lncRNA in cell lines.RESULTS A total of 1 513 transcripts were differentially expressed in Hep-2 and Hep-2/DDP cells,including 399 up-regulated genes and580 down-regulated genes.The major enrichment functions include DNA damage repair,reactive oxygen species,cell death,apoptosis,cell cycle quiescence and epithelial mesenchymal transition.The core nodes of constructing a known molecular interaction network were up-regulated gene vascular endothelial growth factor A（VEGFA）and down-regulated gene protein kinaseB1（PKB1,Also known as AKT1）;four differentially expressed lncRNAs were screened out：MALAT1,LINC01315,AC092757.3 and LINC01822;63 differentially expressed miRNAs were screened.There were 38 miRNAs and 25 miRNAs were up-regulated.The integrated analysis indicated that miRNAs and lncRNAs were widely involved in the multidrug resistance mechanism of cisplatin.Rt-pcr results showed that MALAT1（0.12±0.02）,AC092757.3（0.35±0.02）and MIR155（0.37±0.01）were downregulated in hep-2/DDP cells,Pvalues were 0.036,0.006 and 0.005,respectively.However,MIR34 Awa

关 键 词：喉癌 顺铂 多药耐药 RNA测序 

分 类 号：R739.65[医药卫生—肿瘤]