hsa_circ_0000231调控Wnt/β-catenin信号通路影响舌癌细胞的增殖和迁移  

hsa_circ_0000231 affects the progression of tongue squamous cell carcinoma by activating Wnt/β-catenin signaling pathway

作　　者：陈庆文 王东琴 丁必骁 汤明明[2] 李晓光[3] 周杰玉 徐克[5] 方峥嵘[6] 韩靓[2] 吴昊[1] Chen Qingwen;Wang Dongqin;Ding Bixiao;Tang Mingming;Li Xiaoguang;Zhou Jieyu;Xu Ke;Fang Zhengrong;Han Liang;Wu Hao(Department of Otolaryngology Head and Neck Surgery,Affiliated Hospital of Nantong University,Nantong 226000,China;Department of Otolaryngology Head and Neck Surgery,Affiliated Tumor Hospital of Nantong University,Nantong 226361,China;Department of Maxillofacial Head and Neck Oncology,Shanghai Ninth People′s Hospital,Shanghai Jiaotong University School of Medicine,Shanghai 200011,China;Department of Otolaryngology Head and Neck Surgery,Shanghai Ninth People′s Hospital,Shanghai Jiaotong University School of Medicine,Shanghai 200011,China;Department of Oral and Maxillofacial Surgery,Affiliated Hospital of Nantong University,Nantong 226000,China;Department of Otolaryngology Head and Neck Surgery,Nantong Rich Hospital,Nantong 226010,China)

机构地区：[1]南通大学附属医院耳鼻咽喉头颈外科,南通226000 [2]南通大学附属肿瘤医院头颈外科,南通226361 [3]上海交通大学医学院附属第九人民医院颌面头颈肿瘤科,南通200011 [4]上海交通大学医学院附属第九人民医院耳鼻咽喉头颈外科,南通200011 [5]南通大学附属医院口腔颌面外科,南通226000 [6]南通瑞慈医院耳鼻咽喉头颈外科,南通226010

出　　处：《中华耳鼻咽喉头颈外科杂志》2022年第10期1230-1239,共10页Chinese Journal of Otorhinolaryngology Head and Neck Surgery

基　　金：南通市社会民生科技计划重点项目(MS22018011);南通市科技计划项目(MS12021097);南通市基础科学研究项目(JCZ2022069)。

摘　　要：目的探讨hsa_circ_0000231在舌鳞状细胞癌(tongue squamous cell carcinoma,TSCC)发生、发展中的作用机制。方法收集2014年12月至2017年12月在南通大学附属医院和南通大学附属肿瘤医院收治并进行手术的舌癌患者60例(男32例,女28例,年龄36~84岁),唾液标本来自同时期的10例舌癌患者,以及10名健康志愿者(男5名,女5名,年龄40~75岁),3株舌癌细胞为TSCC常用工具细胞(CAL-27、Tca-8113、HN-4)。采用实时荧光定量反转录聚合酶链反应(quantitative real-time polymerase chain reaction,qRT-PCR)检测60对新鲜配对TSCC组织、10对唾液标本及3株TSCC细胞系中的hsa_circ_0000231的表达;结合随访资料,分析hsa_circ_0000231相对表达量与患者临床病理特征及预后的关系。选择敲低效率最高的细胞行蛋白免疫印迹法(WB)、细胞计数试验(cell counting kit-8,CCK-8)、克隆形成、Transwell侵袭和划痕试验,研究TSCC细胞的增殖、侵袭、转移及上皮间质转化(epithelial-mesenchymal transition,EMT)的能力;使用Wnt/β-catenin通路激活剂LiCl与TSCC细胞共培养,WB检测并研究Wnt/β-catenin信号通路相关蛋白的表达变化。裸鼠成瘤实验比较皮下移植瘤的生长。使用SPSS 25.0统计分析软件行数据分析,组间比较采用t检验和χ^(2)检验。结果hsa_circ_0000231在TSCC患者组织、唾液标本及细胞系CAL-27、Tca-8113和HN-4中高表达,配对癌旁组织、健康人唾液标本及正常人类口腔黏膜细胞(human oral keratinocytes,HOK)中低表达(P值均<0.05)。单因素分析显示hsa_circ_0000231表达水平、肿瘤分化程度和T分级与TSCC患者的生存预后相关(P值均<0.05)。多因素Cox风险回归模型分析显示hsa_circ_0000231表达水平(χ^(2)=5.77,P=0.016)和T分级(χ^(2)=5.27,P=0.029)是TSCC患者预后不良的独立影响因子。WB显示,敲低hsa_circ_0000231表达可以显著提高CAL-27和Tca-8113细胞中EMT相关蛋白即E-钙黏蛋白的表达,并降低Snail、N-钙黏蛋白和波形蛋Objective To explore the action mechanism of hsa_circ_0000231 in the occurrence and development of tongue squamous cell carcinoma(TSCC).Methods Tissue samples of 60 TSCC patients were examined.The patients,including 32 males and 28 females,aged from 36 to 84 years old,underwent surgery in the Affiliated Hospital of Nantong University and Affiliated Tumor Hospital of Nantong University from December 2014 to December 2017.Saliva samples were obtained from healthy volunteers(5 males and 5 females,aged from 40 to 75 years old)and 10 TSCC patients.The TSCC cell lines(CAL-27,Tca-8113 and HN-4)were used.The expression levels of hsa_circ_0000231 in 60 pairs of freshly matched TSCC and para-carcinoma tissue samples,10 pairs of saliva samples and 3 TSCC cell lines were detected by quantitative real-time polymerase chain reaction(qRT-PCR).hsa_circ_0000231 gene interference and lentiviral transfection were constructed,hsa_circ_0000231 in TSCC cell lines CAL-27 and Tca-8113 was knocked down,and the expressions of hsa_circ_0000231 in hsa_circ_0000231 interference group(sh-circ)and no-load lentivirus group(negative control)were tested with qRT-PCR.Cells with the highest knock-down efficiency were selected for CCK-8 test,colony formation assay,transwell invasion assay and scratch assay.The expressions of EMT-related proteins including E-cadherin,snail protein,N-cadherin and vimentin and proteins related to Wnt/β-catenin signaling pathway includingβ-catenin,C-myc,Bcl-2,MMP-9 and Cyclin D1 were measured by western blot.After TSCC cells in the interference group were co-cultured with Wnt/β-catenin pathway activator LiCl,the expressions of above proteins were re-measured by western blot.TSCC cells in interference group and control group were subcutaneously injected into nude mice to compare the effect of hsa_circ_0000231 knockdown on the growths of the tumors grafted subcutaneously in the nude mice.Statistical analysis software 25.0 was used for data analysis,and t-test or chi-square test was used for comparison between groups.Re

关 键 词：舌肿瘤 环状RNA hsacirc0000231 非侵入性 预后 

分 类 号：R739.86[医药卫生—肿瘤]