长链非编码RNA膀胱癌相关转录因子1通过微小RNA-142/自噬相关蛋白7调节自噬对喉癌细胞化疗耐药的影响  

Effect of long non-coding RNA bladder cancer-associated transcript 1 on chemotherapy resistance of laryngeal cancer cells by regulating autophagy through microRNA-142/autophagy-related protein 7

作　　者：张正艳 马文学 齐静静 代红磊 ZHANG Zhengyan;MA Wenxue;QI Jingjing;DAI Honglei(Department of Otolaryngology,JingmenNo.2People's Hospital,Jingmen,Hubei,448000,China)

机构地区：[1]荆门市第二人民医院耳鼻咽喉科,湖北荆门448000

出　　处：《中国耳鼻咽喉头颈外科》2022年第8期483-488,共6页Chinese Archives of Otolaryngology-Head and Neck Surgery

摘　　要：目的 探讨长链非编码RNA(long non-coding RNA,lncRNA)膀胱癌相关转录因子1(bladder cancer-associated transcript 1,BLACAT1)在喉鳞状细胞癌(laryngeal squamous cell carcinoma,LSCC)细胞化疗耐药中的作用。方法 通过顺铂诱导喉癌细胞株Hep-2,建立顺铂耐药喉癌细胞模型(Hep-2/R)。实时荧光定量PCR(RT-qPCR)检测细胞中lncRNA BLACAT1、微小RNA-142(microRNA-142,miR-142)和自噬相关蛋白7(autophagy-related protein 7,ATG7)mRNA的表达;Western blot检测细胞中ATG7、多药耐药蛋白1(multidrug resistance protein 1,MRP1)和微管相关蛋白1轻链3(microtubule associated protein 1 light chain 3,LC3)-II/LC3-I和Beclin 1的表达;MTT实验检测细胞活力。双荧光素酶报告基因实验分别检测BLACAT1和miR-142及miR-142和ATG7之间的靶向作用。结果 Hep-2/R组细胞中lncRNA BLACAT1表达水平(3.58±0.47)显著高于Hep-2组(1.00±0.06),差异有统计学意义(t=9.431,P<0.05)。Hep-2/R组细胞中miR-142表达水平(0.35±0.04)显著低于Hep-2组(1.00±0.05),差异有统计学意义(t=17.583,P<0.05)。与Vector组比较,pcDNA-BLACAT1组Hep-2细胞活力显著升高;与NC-siRNA组比较,BLACAT1-siRNA组Hep-2/R细胞活力显著降低。与WT-BLACAT1和NC mimic共转染组相比,WT-BLACAT1和miR-142共转染的细胞中萤光素酶活性显著降低(t=10.832,P<0.05);与Vector组比较,pcDNA-BLACAT1组细胞中miR-142表达显著降低;与WT-ATG7和NC mimic共转染组相比,WT-ATG7和miR-142共转染的细胞中萤光素酶活性显著降低(t=8.203,P<0.05);与NC mimic组比较,miR-142 mimic组细胞中ATG7蛋白水平均显著降低。使用自噬抑制剂3-MA处理Hep-2细胞后,pcDNA-BLACAT1显著促进自噬蛋白ATG7、LC3-II/LC3-I和Beclin 1的表达,miR-142 mimic处理后自噬蛋白水平降低。使用DDP处理Hep-2/R细胞后,BLACAT1-siRNA显著抑制Hep-2/R细胞活力和MRP1蛋白的表达,而miR-142 inhibitor逆转这一结果。结论 LncRNA BLACAT1通过miR-142/ATG7信号通路促进LSCC细胞的自噬和化疗耐药性。OBJECTIVE To investigate the role of long non-coding RNA (lncRNA) bladder cancer-associated transcript 1(BLACAT1) in chemoresistance of laryngeal squamous cell carcinoma(LSCC) cells.METHODS A cisplatin-resistant laryngeal cancer cell model(Hep-2/R) was established by inducing the laryngeal cancer cell line Hep-2 with cisplatin.Real-time quantitative PCR(RT-qPCR) experiments were used to detect the mRNA expressions of lncRNA BLACAT1,microRNA-142(miR-142) and autophagy-related protein 7(ATG7) in cells.Western blot was used to detect the expression of ATG7,multidrug resistant protein 1(MRP1),microtubule associated protein 1 light chain 3(LC3)-II/LC3-I and Beclin 1 in cells.Cell viability was detected by MTT assay.Dual luciferase reporter assay was used to detect the targeting effect between BLACAT1 and miR-142,and miR-142 and ATG7,respectively.RESULTS The expression level of lncRNA BLACAT1(3.58±0.47) in cells of Hep-2/R group was significantly higher than that of Hep-2 group(1.00±0.06),and the difference was statistically significant(t=9.431,P<0.05).The expression level of miR-142 (0.35±0.04) in Hep-2/R group was significantly lower than that in Hep-2 group(1.00±0.05),and the difference was statistically significant(t=17.583,P<0.05).Compared with the Vector group,the viability of Hep-2 cells was significantly increased in the pcDNA-BLACAT1 group;Compared with the NC-siRNA group,the viability of the Hep-2/R cells was significantly decreased in the BLACAT1-siRNA group.Compared with the WT-BLACAT1 and NC mimic co-transfection group,the luciferase activity was significantly reduced in cells co-transfected with WT-BLACAT1 and miR-142(t=10.832,P<0.05).Compared with the Vector group,the expression of miR-142 was significantly decreased in the pcDNA-BLACAT1 group.Compared with the WT-ATG7 and NC mimic co-transfection group,the luciferase activity was significantly reduced in cells co-transfected with WT-ATG7 and miR-142(t=8.203,P<0.05).Compared with the NC mimic group,the protein levels of ATG7 was significantly decreas

关 键 词：喉肿瘤 耐药性 药物耐受性 长链非编码RNA膀胱癌相关转录因子1 微小RNA-142 自噬 

分 类 号：R739.65[医药卫生—肿瘤]