lncRNA CASC11通过EZH2调控PI3K/AKT通路促进食管鳞癌细胞顺铂耐药的机制研究  

Study on the mechanism of lncRNA CASC11 regulating PI3K/AKT pathway through EZH2 to promote the cisplatin resistance in esophageal squamous cell carcinoma cells

作　　者：王伟[1] 何天乐 李扬[3] 杨素清 WANG Wei;HE Tianle;LI Yang;YANG Suqing(Department of Thoracic Surgery,the First Hospital of Handan City,Hebei Handan 056000,China;Naval Medical University of PLA,Shanghai 200433,China;Department of Sensory Control,the Second Hospital of Hebei Medical University,Hebei Shijiazhuang 050000,China)

机构地区：[1]邯郸市第一医院胸外科,河北邯郸056000 [2]中国人民解放军海军军医大学,上海200433 [3]河北医科大学第二医院感控科,河北石家庄050000

出　　处：《现代肿瘤医学》2023年第14期2624-2631,共8页Journal of Modern Oncology

基　　金：2022年度河北省医学科学研究课题计划(编号:20220981)。

摘　　要：目的:探讨lncRNA CASC11对EZH2及磷脂酰肌醇-3-激酶(PI3K)/蛋白激酶B(AKT)信号轴的调控作用,及对食管鳞状细胞癌(ESCC)细胞顺铂(DDP)耐药性的影响。方法:取ESCC组织(76例)及癌旁组织(76例),并体外培养正常食管黏膜上皮细胞(HET-1a)及ESCC细胞系(TE-1、Eca109、KYSE150、KYSE450和KYSE510),qRT-PCR法检测lncRNA CASC11表达。逐步增加DDP浓度构建DDP耐药ESCC细胞(TE-1/DDP),并随机分成si-NC组、si-lncRNA-CASC11组、si-lncRNA CASC11+IGF-1组、si-lncRNA CASC11+si-PTEN组,另取正常培养的TE-1细胞为Control组。克隆形成实验、CCK-8、流式细胞术、Transwell分别检测细胞增殖、耐药性、凋亡、迁移;qRT-PCR及Western Blot法检测lncRNA CASC11及EZH2、PI3K/AKT途径抑制物PTEN、PI3K/AKT通路蛋白、EMT标记蛋白(E-cadherin、N-cadherin)表达;RNA免疫沉淀(RIP)法检测PTEN对EZH2的调控关系;染色质免疫沉淀(ChIP)检测EZH2、PTEN、lncRNA CASC11三者间调控关系。将lncRNA CASC11敲低TE-1/DDP细胞接种于裸鼠皮下并进行DDP干预,检测瘤体体积及瘤体重量,免疫组化分析Ki67活性。结果:lncRNA CASC11在ESCC癌组织、细胞系和TE-1/DDP细胞中的表达均显著升高(P<0.05)。敲低lncRNA CASC11可抑制TE-1/DDP细胞增殖、迁移及EMT进程,触发细胞凋亡,并抑制细胞耐药及EZH2-PI3K/AKT生存途径的活化(P<0.05)。裸鼠荷瘤实验证实敲低lncRNA CASC11可增加TE-1细胞对DDP的敏感性(P<0.05)。EZH2可结合PTEN启动子并降低PTEN表达,而敲低lncRNA CASC11可减少EZH2与PTEN启动子区域的结合。PTEN敲低或给予IGF-1干预,均可部分逆转lncRNA CASC11敲低发挥的抗癌及抗DDP耐药性产生的作用(P<0.05)。结论:lncRNA CASC11可通过与EZH2相互作用来沉默PTEN,激活PI3K/AKT介导的细胞存活途径,提高ESCC对DDP的耐药性,而敲低lncRNA CASC11可降低癌细胞对DDP的耐药性,增强ESCC对DDP的化疗敏感性。Objective:To investigate the regulation of lncRNA CASC11 on the EZH2 and phosphatidylinositol-3-kinase(PI3K)/protein kinase B(AKT)signal axis,and its influence on the resistance of esophageal squamous cell carcinoma(ESCC)cells to cisplatin(DDP).Methods:ESCC tissue(76 cases)and adjacent tissue(76 cases)were taken.The normal esophageal mucosal epithelial cells(HET-1a)and ESCC cell lines(TE-1,Eca109,KYSE150,KYSE450 and KYSE510)were cultured in vitro,and the expression of lncRNA CASC11 was detected by qRT-PCR.DDP resistant ESCC cells(TE-1/DDP)were constructed by gradually increasing DDP concentration,and randomly divided into si-NC group,si-lncRNA CASC11 group,si-lncRNA CASC11+IGF-1 group,and si-lncRNA CASC11+si-PTEN group,and normal cultured TE-1 cells were also taken as Control group.Cell proliferation,drug resistance,apoptosis and migration were detected by clonal formation assay,CCK-8,flow cytometry and Transwell,respectively.qRT-PCR and Western Blot method were used to detect the expression of lncRNA CASC11 and EZH2,PI3K/AKT pathway inhibitor PTEN,PI3K/AKT pathway proteins,EMT marker proteins(E-cadherin,N-cadherin).RNA immunoprecipitation(RIP)method was used to detect the regulatory relationship of PTEN to EZH2.Chromatin immunoprecipitation(ChIP)was used to detect the regulatory relationship among EZH2,PTEN,and lncRNA CASC11.TE-1/DDP cells knocked down by lncRNA CASC11 were inoculated subcutaneously in nude mice.The tumor volume and tumor weight were detected after DDP intervention,and Ki67 activity was analyzed by immunohistochemistry.Results:The expression of lncRNA CASC11 was significantly increased in ESCC cancer tissues,ESCC cancer cell lines and TE-1/DDP cells(P<0.05).Knockdown of lncRNA CASC11 was able to inhibit cancer cell proliferation,migration,and EMT process,trigger cell apoptosis,and inhibit cell resistance and the activation of EZH2-PI3K/AKT survival pathway(P<0.05).The nude mouse tumor-bearing test confirmed that knockdown of lncRNA CASC11 was able to increase the sensitivity of TE-1 cells to DDP

关 键 词：长链非编码RNA CASC11 Zeste增强子同源物2 磷脂酰肌醇-3-激酶 蛋白激酶B 顺铂耐药性 

分 类 号：R735.1[医药卫生—肿瘤]