摘要
目的:探讨长链非编码RNA(lncRNA)LINC01426对非小细胞肺癌(NSCLC)顺铂(DDP)耐药的影响,并研究其潜在机制。方法:体外培养A549细胞和抗DDP细胞株A549/DDP,逆转录定量聚合酶链反应(RT-qPCR)检测细胞中LINC01426、zeste增强子同源物2(EZH2)mRNA表达;LINC01426和EZH2之间的相互作用通过RNA-蛋白质相互作用预测(RIP-seq)、RNA免疫沉淀(RIP)、RT-qPCR和染色质免疫沉淀(ChIP)测定进行验证。将A549/DDP细胞分为对照组(Control组)、si-NC组、si-LINC01426组、si-LINC01426+pc-NC组、si-LINC01426+pc-EZH2组,转染培养48 h,用RT-qPCR检测各组细胞中LINC01426、EZH2 mRNA、PTEN mRNA表达,CCK-8检测细胞对DDP的敏感性,集落形成实验检测细胞增殖,流式细胞术检测细胞凋亡,蛋白质印迹(Western blot)分析细胞中EZH2、PTEN、AKT、p-AKT蛋白表达。裸鼠移植瘤模型验证LINC01426是否参与体内NSCLC细胞对DDP的敏感性。结果:A549/DDP细胞中LINC01426、EZH2 mRNA水平高于A549细胞;LINC01426主要分布在细胞核中,能够与EZH2相互作用;敲低LINC01426可上调PTEN表达,降低p-AKT/AKT比值,增强A549/DDP细胞对DDP的敏感性,抑制A549/DDP细胞增殖,并诱导细胞凋亡(均P<0.05);裸鼠移植瘤实验证实敲低LINC01426可上调PTEN表达,降低p-AKT/AKT比值,抑制A549/DDP细胞体内肿瘤生长并增强体内A549/DDP细胞对DDP的敏感性(P<0.05),沉默PTEN可减弱敲低LINC01426对裸鼠体内A549/DDP细胞DDP敏感性的影响(P<0.05);过表达EZH2可逆转LINC01426敲低对细胞增殖的抑制和对细胞凋亡、DDP敏感性的促进作用(P<0.05)。结论:LINC01426在A549/DDP细胞中上调,敲低LINC01426可能通过抑制EZH2来上调PTEN表达,进而抑制PI3K/AKT通路的活化,抑制A549/DDP细胞体外增殖和体内肿瘤生长并增强NSCLC细胞对DDP的敏感性。
Objective:To investigate the effect of long non-coding RNA(lncRNA)LINC01426 on cisplatin(DDP)resistance in non-small cell lung cancer(NSCLC)and its potential mechanism.Methods:A549 cells and anti-DDP cell line A549/DDP were cultured in vitro.The reverse transcription-quantitative polymerase chain reaction(RT-qPCR)was performed to detect the mRNA expressions of LINC01426 and enhancer of zeste homolog 2(EZH2)in cells.The interaction between LINC01426 and EZH2 was validated by RNA-protein interaction prediction(RIP-seq),RNA immunoprecipitation(RIP),RT-qPCR and chromatin immunoprecipitation(ChIP)assays.A549/DDP cells were grouped into Control group,si-NC group,si-LINC01426 group,si-LINC01426+pc-NC group,and si-LINC01426+pc-EZH2 group,and were transfected and cultured for 48 h.RT-qPCR was implemented to detect LINC01426,EZH2 and PTEN mRNA expression of cells in each group.CCK-8 was implemented to detect the sensitivity of cells to DDP.Colony formation assay was used to detect cell proliferation.Flow cytometry was used to detect cell apoptosis.Western blot was performed to analyze EZH2,PTEN,AKT and p-AKT protein expression in cells.A nude mouse xenograft model was performed to verify whether LINC01426 was involved in the sensitivity of NSCLC cells to DDP in vivo.Results:The mRNA levels of LINC01426 and EZH2 in A549/DDP cells were higher than those in A549 cells.LINC01426 was mainly distributed in the nucleus and could interact with EZH2.The knockdown of LINC01426 upregulated PTEN expression,reduced p-AKT/AKT ratio,enhanced the sensitivity of A549/DDP cells to DDP,inhibited the proliferation of A549/DDP cells,and induced apoptosis(all P<0.05).The nude mouse xenograft experiments confirmed that knockdown of LINC01426 upregulated PTEN expression,decreased p-AKT/AKT ratio,inhibited the growth of A549/DDP cells in vivo and enhanced the sensitivity of A549/DDP cells to DDP in vivo(P<0.05).Silencing PTEN attenuated the effect of LINC01426 knockdown on DDP sensitivity of A549/DDP cells in nude mice(P<0.05).Overexpression of EZH2 could reverse the inhibition of LINC01426 knockdown on cell proliferation and the promotion on apoptosis and DDP sensitivity(P<0.05).Conclusion:LINC01426 is up-regulated in A549/DDP cells,and knockdown of LINC01426 may upregulate the expression of PTEN by inhibiting EZH2,thereby inhibiting the activation of PI3K/AKT pathway,inhibiting A549/DDP cell proliferation in vitro and tumor growth in vivo and enhancing the sensitivity of NSCLC cells to DDP.
作者
陈云峰
崔文洁
施海
刘向群
CHEN Yunfeng;CUI Wenjie;SHI Hai;LIU Xiangqun(Department of Respiratory and Critical Care Medicine,Xuzhou First People's Hospital,Jiangsu Xuzhou 221116,China)
出处
《现代肿瘤医学》
CAS
北大核心
2023年第14期2605-2613,共9页
Journal of Modern Oncology
基金
徐州市卫生健康委面上项目(编号:XWKYHT20210529)
徐州市第一人民医院青苗工程(编号:QMHB2021017)。
