微小RNA-33b-5p靶向ETS1在胰腺癌吉西他滨耐药中的作用  

MicroRNA-33b-5p regulates the resistance of pancreatic cancer cells to gemcitabine chemotherapy by targeting ETS1

作　　者：李文磊 付强[2] 刘攀 张旭 罗乾坤 禹鹏飞 秦涛[2] Li Wenlei;Fu Qiang;Liu Pan;Zhang Xu;Luo Qiankun;Yu Pengfei;Qin Tao(Department of Hepatobiliary and Pancreatic Surgery,People’s Hospital of Zhengzhou University,Zhengzhou 450003,China;Department of Hepatobiliary and Pancreatic Surgery,Henan Provincial People’s Hospital,Zhengzhou 450003,China)

机构地区：[1]郑州大学人民医院肝胆胰腺外科,郑州450003 [2]河南省人民医院肝胆胰腺外科,郑州450003

出　　处：《中华实验外科杂志》2023年第2期262-265,共4页Chinese Journal of Experimental Surgery

基　　金：河南省省部共建项目(SB201901083);河南省科技攻关项目(212102310151)。

摘　　要：目的探讨微小RNA-33b-5p(miR-33b-5p)靶向E26转录因子-1(E26 transformation specific-1,ETS1)在胰腺导管腺癌(PDAC)吉西他滨耐药中的作用。方法通过实时荧光定量聚合酶链反应(RT-qPCR)和蛋白质印迹法(Western blot)检测胰腺癌细胞ASPC-1和胰腺癌耐药细胞ASPC-1/GEM中miR-33b-5p和ETS1的表达水平,双荧光素酶报告实验验证miR-33b-5p与ETS1之间的靶向关系;免疫组织化学染色检测PDAC患者敏感组与抵抗组ETS1的表达;转染ASPC-1/GEM细胞,分为NC mimic组、miR-33b-5p mimic组、ETS1小干扰RNA(siRNA)NC组及ETS1 siRNA组,RT-qPCR检测转染后miR-33b-5p、ETS1 mRNA的表达水平,Western blot检测ETS1的表达水平,细胞计数试剂盒(CCK-8)检测细胞活性并计算吉西他滨半数抑制浓度(IC_(50)),流式细胞术检测细胞凋亡水平。结果与ASPC-1细胞比较,ASPC-1/GEM细胞株中miR-33b-5p表达下降(t=-11.011,P<0.05)、ETS1表达升高(P<0.05);双荧光素酶报告实验表明miR-33b-5p可明显降低野生型ETS1-33b-5p-WT载体荧光素酶活性(t=-4.453,P<0.05);免疫组织化学结果显示ETS1在抵抗组中表达量明显高于敏感组(8.11±2.80比2.78±0.83,P<0.05);miR-33b-5p mimic组IC_(50)明显低于NC mimic组[(25.10±1.77)μmol/L比(43.63±3.04)μmol/L,t=-9.110,P<0.05],miR-33b-5p表达量明显高于NC mimic组(t=19.444,P<0.05),ETS1的表达量明显低于NC mimic组(P<0.05),凋亡率明显高于NC mimic组[(18.8±1.9)%比(5.3±0.4)%,t=12.239,P<0.05];ETS1 siRNA组吉西他滨IC_(50)明显低于ETS1 siRNA NC组[(20.39±0.89)μmol/L比(37.38±2.62)μmol/L,t=-10.628,P<0.05]。结论miR-33b-5p可能通过抑制ETS1表达促进PDAC对吉西他滨化疗的敏感性。Objective To investigate the role of MicroRNA-33b-5p(mi-R33b-5p)targeting E26 transformation Specification-1(ETS1)in gemcitabine resistance to pancreatic ductal adenocarcinoma(PDAC).Methods The expression levels of miR-33b-5p and ETS1 in pancreatic cancer cells ASPC-1 and drug-resistant pancreatic cancer cells ASPC-1/GEM were detected by real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)and Western blotting.The targeting relationship between miR-33b-5p and ETS1 was verified by double luciferase report experiment.The expression of ETS1 in sensitive group and resistant group of PDAC patients was detected by immunohistochemical staining.Transfected ASPC-1/GEM cells were divided into NC mimic group,miR-33b-5p mimic group,ETS1 small interfering RNA(siRNA)NC group and ETS1 siRNA group.The expression level of miR-33b-5p and ETS1 mRNA after transfection was detected by RT-qPCR.The expression level of ETS1 was detected by Western blotting.The cell activity was detected by cell counting kit-8(CCK-8)assay,and the semi inhibitory concentration of gemcitabine was calculated.The cell apoptosis was detected by flow cytometry.Results Compared with ASPC-1 cells,the expression of miR-33b-5p in ASPC-1/GEM cells was decreased(t=11.011,P<0.05),and the expression of ETS1 was increased(P<0.05).Double luciferase reporting experiment showed that miR-33b-5p could significantly reduce the luciferase activity of wild-type ETS1-33b-5p-WT vector(t=-4.453,P<0.05).Immunohistochemical results showed that the expression level of ETS1 in resistant group was significantly higher than that in sensitive group(8.11±2.80 vs.2.78±0.83,P<0.05).The half maximal inhibitory concentration(IC_(50))of miR-33b-5p mimic group was significantly lower than that of NC mimic group[(25.10±1.77)μmol/L vs.(43.63±3.04)μmol/L,t=-9.110,P<0.05].The expression of miR-33b-5p was significantly increased(t=19.444,P<0.05),the expression of ETS1 was significantly decreased(P<0.05),and the apoptosis rate was significantly increased[(18.8±1.9)%vs.(5.3±0

关 键 词：胰腺癌 吉西他滨 耐药 

分 类 号：R735.9[医药卫生—肿瘤]