EB病毒诱导基因3增强结肠癌5-氟尿嘧啶耐药株耐药性的研究  

Study on Epstein-Barr virus-induced gene 3 enhances the drug resistance of 5-fluorouracil resistant colorectal cancer cells

作　　者：梁艳芳 叶子瑜 马燕 林碧华 陈灿 曾今诚 LIANG Yanfang;YE Ziyu;MA Yan;LIN Bihua;CHEN Can;ZENG Jincheng(Department of Pathology,Dongguan Hospital Affiliated to Jinan University/Marina Bay Central Hospitalof Dongguan,Dongguan,Guangdong 523905,China;Dongguan Key Laboratory of Medical Bioactive Molecular Developmental and Translational Research,Guangdong Medical University,Dongguan,Guangdong 523808,China;Guangdong Provincial Key Laboratory of Medical Molecular Diagnostics,Guangdong Medical University,Dongguan,Guangdong 523808,China)

机构地区：[1]广东省东莞市滨海湾中心医院/暨南大学附属东莞医院病理科,523905 [2]广东医科大学东莞市医学活性分子开发与转化重点实验室,广东东莞523808 [3]广东医科大学广东省医学分子诊断重点实验室,广东东莞523808

出　　处：《重庆医学》2020年第13期2082-2086,共5页Chongqing medicine

基　　金：东莞市社会科技发展(一般)项目(2018507150251294);广东省医学科研基金项目(A2018123);广东省基础与应用基础研究基金联合基金(粤莞)项目(2019A1515110042)。

摘　　要：目的建立结肠癌(CRC)5-氟尿嘧啶(5-Fu)耐药株探讨EB病毒诱导基因3(EBI3)与CRC 5-Fu耐药的关系。方法采用5-Fu持续接触浓度递增诱导法建立人CRC耐药细胞系;CCK-8法检测细胞增殖和耐药性;4′,6-二脒基-2-苯基吲哚(DAPI)染色观察细胞核形态学改变;流式细胞术检测细胞周期及细胞凋亡;EBI3 CRISPR激活质粒和EBI3 CRISPR/Cas9敲除质粒转染结肠癌耐药和非耐药细胞后,Western blot检测EBI3、细胞色素P450家族成员1B1(CYP1B1)和有丝分裂原活化蛋白激酶激酶激酶2(MEKK2)表达。结果采用5-Fu持续接触浓度递增诱导法建立人结肠CRC细胞系SW1116/5-Fu和LoVo/5-Fu;细胞周期检测结果显示,与SW1116和LoVo细胞相比,SW1116和SW1116/5-Fu细胞周期各期百分比无明显差异(P>0.05)。与LoVo细胞相比,LoVo/5-Fu细胞G1期细胞百分比明显增高(P<0.05),S期细胞百分比明显降低(P<0.05);Western blot检测结果显示,与SW1116和LoVo细胞相比,SW1116/5-Fu和LoVo/5-Fu细胞EBI3、CYP1B1和MEKK2表达均明显增高;SW1116、SW1116/5-Fu、LoVo和LoVo/5-Fu细胞转染EBI3敲除质粒后,5-Fu作用细胞的半抑制浓度(IC50)值明显降低(P<0.05),而细胞转染EBI3激活质粒后,5-Fu作用细胞的IC50值明显增高(P<0.05)。结论EBI3可能介导了CRC细胞对5-Fu的耐药过程。Objective To explore the relationship between Epstein-Barr virus-induced gene 3(EBI3)and the drug resistance of 5-Fluorouracil(5-Fu)in colorectal cancer cells via establishing 5-Fu drug-resistant colon cancer strains.Methods The human colorectal cancer drug-resistant cellswere established by continuously exposing the colorectal cancercells to ascending doses of 5-Fu.Cell proliferation and drug resistance were detected by CCK-8.Changes in nuclear morphology was observed by 4′,6-diamidino-2-phenylindole(DAPI)staining.Cell cycle was detected by using flow cytometry.APC Annexin V-7AAD double staining was used to detect apoptosis.EBI3 CRISPR activation plasmid and EBI3 CRISPR/Cas9 knockout plasmid were transfected into colorectal cancer resistant and non-resistant cells,and Western blot was used to detect EBI3,CYP1B1 and MEKK2 expression.Results The human colorectal cancer 5-Furesistant cell lines SW1116/5-Fu and LoVo/5-Fu were successfully constructed after continuous exposure to increased concentration of 5-Fu.The cell cycle test results showed that there was no significant difference in the percentages of cell cycle stages between SW1116 and SW1116/5-Fu cells(P>0.05).However,compared with LoVo cells,the percentage of cells in the G1 phase of LoVo/5-Fu cells was significantly increased(P<0.05),and the percentage of cells in the S phase was significantly decreased(P<0.05).Western blot showed that,compared with SW1116 and LoVo cells,the expression of EBI3,CYP1B1 and MEKK2 in SW1116/5-Fu and LoVo/5-Fu cells were significantly increased.The IC50 values of 5-Fu were significantly decreased after SW1116,SW1116/5-Fu,LoVo and LoVo/5-Fu cellstransfected with EBI3 knockout plasmid(P<0.05),and the IC50 value of 5-Fuwas significantly increased after cells transfected with EBI3 activating plasmid(P<0.05).Conclusion EBI3 may mediate the resistance of colorectal cancer cells to 5-Fu.

关 键 词：EB病毒诱导基因3 结肠肿瘤 5-氟尿嘧啶 耐药细胞系 

分 类 号：R735.3[医药卫生—肿瘤]