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

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(P<0.05).EZH2 was able to bind to the PTEN promoter and reduce the expression of PTEN,while knockdown of lncRNA CASC11 was able to reduce the binding of EZH2 to the PTEN promoter region.PTEN knockdown or IGF-1 intervention was able to partially reverse the effects of lncRNA CASC11 knockdown on anti-cancer and anti-DDP resistance(P<0.05).Conclusion:lncRNA CASC11 can interact with EZH2 to silence PTEN,activate PI3K/AKT-mediated cell survival pathways,and increase ESCC resistance to DDP.The knockdown of lncRNA CASC11 can reduce the resistance of cancer cells to DDP and enhance the sensitivity of ESCC to DDP chemotherapy.