miR-216b-5p靶向ATG5介导自噬逆转食管癌Eca109细胞的顺铂耐药性  

miR-216b-5p mediates autophagy to reverses cisplatin resistance of esophageal cancer Eca109 cells by targeting ATG5

作　　者：邱善婷 李晓燕 陈哲聪 高梦源 金舒怡 陈文虎 QIU Shanting;LI Xiaoyan;CHEN Zhecong;GAO Mengyuan;JIN Shuyi;CHEN Wenhu(School of Basic Medical Sciences&Forensic Medicine,Hangzhou Medical College,Hangzhou 310059,Zhejiang,China)

机构地区：[1]杭州医学院基础医学与法医学院,浙江杭州310059

出　　处：《中国肿瘤生物治疗杂志》2023年第7期552-559,共8页Chinese Journal of Cancer Biotherapy

基　　金：浙江省中医药科技计划项目(No.2021ZB078);浙江省大学生创新创业项目(No.202113023001);浙江省医药卫生科技计划项目(No.2022RC124);杭州医学院基本科研业务费项目(No.KYQN202001,No.KYYB202003)。

摘　　要：目的:探讨miR-216b-5p对食管癌Eca109细胞顺铂(DDP)耐药性的影响及其作用机制。方法:采用qPCR法检测miR-216b-5p在食管癌细胞TE-1、KYSE-150、Eca109和耐药细胞Eca109/DDP中的表达水平。利用脂质体转染技术分别将miR-216b-5p mimic及mimic NC、自噬相关蛋白5(ATG5)过表达质粒转染到Eca109/DDP细胞中,用CCK-8、EdU法和FCM分别检测转染后细胞的增殖和凋亡;mRFP-eGFP-LC3双荧光标记实验检测mRFP-eGFP-LC3慢病毒感染后各组细胞自噬发生情况,WB法检测自噬相关蛋白LC3、Beclin 1和P62表达。用荧光素酶报告基因实验验证miR-216b-5p与ATG5的靶向关系,WB法检测ATG5的表达。建立裸鼠Eca109/DDP细胞移植瘤模型,观察miR-216b-5p过表达对移植瘤生长的影响。结果:miR-216b-5p在TE-1、KYSE-150、Eca109和Eca109/DDP细胞中均呈低表达(均P<0.05)。过表达miR-216b-5p可显著抑制Eca109/DDP细胞的增殖并诱导凋亡(均P<0.05),减少细胞中自噬小体数量(P<0.05),下调LC3Ⅱ/LC3Ⅰ比值和Beclin 1蛋白水平、上调P62蛋白水平(均P<0.05)。双荧光素酶报告基因实验证实miR-216b-5p靶向并负调控ATG5的表达(P<0.05),过表达ATG5可使miR-216b-5p mimic对Eca109/DDP细胞增殖、自噬的抑制作用和凋亡的诱导作用明显减弱(均P<0.05),自噬相关蛋白P62表达降低、LC3Ⅱ/LC3Ⅰ比值和Beclin 1表达升高(均P<0.05)。荷瘤实验结果表明,miR-216b-5p过表达可显著抑制裸鼠移植瘤的生长(P<0.05)。结论:miR-216b-5p过表达可逆转食管癌Eca109/DDP细胞对DDP的耐药性,其机制可能与靶向负调控ATG5表达并影响细胞自噬有关。Objective:To investigate the effect of miR-216b-5p on cisplatin(DDP)resistance in esophageal cancer Eca109 cells and its mechanism.Methods:The expression levels of miR-216b-5p in esophageal cancer cells TE-1,KYSE-150,Eca109 and drug resistant Eca109/DDP cells were detected by qPCR.The miR-216b-5p mimics,mimic NC and autophagy related protein 5(ATG5)over-expressed plasmids were transfected into Eca109/DDP cells by liposome transfection technique.The proliferation and apoptosis of transfected Eca109/DDP cells were detected by CCK-8,EdU methods and flow cytometry,respectively.The occurrence of autophagy in each group of cells after transfection with mRFP-eGFP-LC3 lentivirus was examined by mRFP-eGFP-LC3 dual fluorescent labeling assay,and the expression of autophagy-related markers,LC3,Beclin 1 and P62,was detected by WB.The targeting relationship between miR-216b-5p and ATG5 was verified by luciferase reporter gene assay,and the expression of ATG5 was detected by Western blotting.A nude mouse Eca109/DDP cell transplanted tumor model was established to observe the effect of miR-216b-5p over-expression on the growth of transplanted tumor.Results:miR-216b-5p was lowly expressed in TE-1,KYSE-150,Eca109 and Eca109/DDP cells(all P<0.05).Over-expression of miR-216b-5p significantly inhibited the proliferation and induced apoptosis of Eca109/DDP cells(both P<0.05),reduced the number of autophagosomes in transfected cells(P<0.05),and down-regulated LC3Ⅱ/LC3Ⅰratio and Beclin 1 protein level,but up-regulated P62 protein level(all P<0.05).Luciferase reporter gene assay confirmed that miR-216b-5p negatively regulated ATG5 expression(P<0.05).Over-expression of ATG5 could significantly weaken the effects of miR-216b-5p mimic on suppressing proliferation,autophagy and inducing apoptosis of Eca109/DDP cells(all P<0.05),and reduce the expression of autophagy related protein P62,but increase the LC3Ⅱ/LC3Ⅰratio and Beclin 1 protein level(all P<0.05).The tumor bearing experiments showed that over-expression of miR-216b-5p could

关 键 词：食管癌 Eca109/DDP细胞 miR-216b-5p 自噬 增殖 凋亡 顺铂耐药 

分 类 号：R735.1[医药卫生—肿瘤] R730.2[医药卫生—临床医学]