摘要
目的探讨长链非编码RNA RP11-159K7.2在鼻腔鼻窦鳞状细胞癌(sinonasal squamous cell carcinoma,SNSCC)进展中的作用与机制。方法选取2009—2014年哈尔滨医科大学附属第二医院耳鼻咽喉头颈外科SNSCC手术患者的癌组织和癌旁非癌组织标本65例,通过RNAscope原位杂交技术检测RP11-159K7.2在SNSCC组织及癌旁组织中的表达,观察其与预后的关系。运用规律成簇间隔短回文重复序列(CRISPR)/CRISPR相关蛋白9(CRISPR/Cas9)方法敲减RPMI-2650细胞(SNSCC细胞系)RP11-159K7.2的表达。体外实验分别用细胞增殖试剂盒8(CCK-8)增殖实验、划痕实验和Transwell实验等观察下调RP11-159K7.2后,SNSCC细胞增殖、迁移和侵袭等生物学行为的变化。体内实验观察RP11-159K7.2下调后,SNSCC裸鼠移植瘤生长的改变。机制研究运用蛋白质芯片方法,筛选RP11-159K7.2可能结合的蛋白质,运用蛋白免疫印迹法(WB)和RNA免疫共沉淀技术鉴定RP11-159K7.2结合的蛋白质。采用SPSS 17.0软件进行统计学处理。结果RP11-159K7.2在SNSCC组织中的表达显著高于癌旁组织,与T分级、淋巴结转移及分化程度密切相关(χ^(²)值分别为4.697、4.235和10.753,P值均<0.05)。RP11-159K7.2高表达患者的5年生存率明显低于RP11-159K7.2低表达患者(P=0.0137)。RP11-159K7.2下调后,SNSCC细胞的增殖、迁移及侵袭能力显著下降,SNSCC移植瘤生长受到明显抑制。RP11-159K7.2可能结合的蛋白质有31种,其可直接与核因子-κB(NF-κB)蛋白结合,对SNSCC细胞增殖和侵袭的调控依赖NF-κB。结论RP11-159K7.2在SNSCC中表达增高,有望成为SNSCC预后评估的潜在分子标志物。其在SNSCC中的作用机制与结合和调控NF-κB蛋白有关。
Objective To explore the role and mechanism of long non-coding RNA RP11-159K7.2 in the progression of sinonasal squamous cell carcinoma(SNSCC).Methods Sixty-five cases of SNSCC tissues and adjacent tissues were selected from the Department of Otorhinolaryngology Head and Neck Surgery,the Second Affiliated Hospital of Harbin Medical University from 2009 to 2014.The expression of RP11-159K7.2 in SNSCC and adjacent tissues was detected by RNAscope in situ hybridization to observe its association with prognosis.Clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR associated proteins 9(CRISPR/Cas9)was used to knockout the expression of RP11-159K7.2 in RPMI-2650 cells(SNSCC cell line).Cell counting kit-8(CCK-8),wound healing and Transwell were performed to observe the changes of proliferation,migration and invasion of SNSCC cells in vitro after down-regulation of RP11-159K7.2.Moreover,the growth of xenograft in nude mice after down-regulation of RP11-159K7.2 was examined in vivo.Mechanically,the protein chip,Western blot and RNA immunoprecipitation were performed to identify the proteins bound by RP11-159K7.2.SPSS 17.0 was used for statistical analysis.Results The expression of RP11-159K7.2 in SNSCC tissue was significantly higher than that in adjacent tissues.RP11-159K7.2 expression was closely related with T grade,nodal metastasis and differentiation of SNSCC(χ^(2) value was 4.697,4.235 and 10.753,respectively,all P<0.05).The five-year survival rate of RP11-159K7.2 high expression patients was significantly lower than that of RP11-159K7.2 low expression ones(P=0.0137).After the down-regulation of RP11-159K7.2,the proliferation,migration and invasion ability of SNSCC cells decreased significantly,and the growth of SNSCC xenograft was significantly inhibited.There were 31 candidate proteins that may bind to RP11-159K7.2.RP11-159K7.2 directly bound to nuclear factor-κB(NF-κB)in SNSCC cells,and the regulation of RP11-159K7.2 on the proliferation and invasion of SNSCC cells depended on NF-κB.Conclu
作者
王鹏
于博宇
王馨
王婧婷
李秋影
孙亚男
田霖丽
刘鸣
Wang Peng;Yu Boyu;Wang Xin;Wang Jingting;Li Qiuying;Sun Yanan;Tian Linli;Liu Ming(Department of Otorhinolaryngology Head and Neck Surgery,the Second Affiliated Hospital of Harbin Medical University,Harbin 150001,China)
出处
《中华耳鼻咽喉头颈外科杂志》
CSCD
北大核心
2021年第6期626-634,共9页
Chinese Journal of Otorhinolaryngology Head and Neck Surgery
基金
国家自然科学基金(81272965,81772874)
中国博士后科学基金(2020M670926)。
