MiR-200c通过调控糖酵解逆转胃癌细胞紫杉醇耐药的实验研究  

Experimental study on MiR-200c reversing paclitaxel resistance in gastric cancer cells by regulating glycolysis

作　　者：周欣亮[1] 李艳艳[1] 张雪[1] 施明亮 赵瑞撵[2] 薛永娴 张璁[2] ZHOU Xinliang;LI Yanyan;ZHANG Xue;SHI Mingliang;ZHAO Ruinian;XUE Yongxian;ZHANG Cong(Fourth Hospital of Hebei Medical University,Shijiashuang,Hebei 05001l,China;Departmentof Oncology,Jinan People's Hospital,Jinan,Shandong 271100,China)

机构地区：[1]河北医科大学第四医院肿瘤内科,河北石家庄050011 [2]河北医科大学第四医院科研中心,河北石家庄050011 [3]济南市人民医院肿瘤科,山东济南271100

出　　处：《中华肿瘤防治杂志》2024年第22期1370-1377,共8页Chinese Journal of Cancer Prevention and Treatment

基　　金：河北省自然科学基金青年科学基金(H2020206435);河北省卫健委青年科技课题(20211231)。

摘　　要：目的探讨miR-200c在逆转胃癌细胞紫杉醇耐药中的作用及其机制。方法构建紫杉醇耐药的胃癌细胞株(AGS/PTX和HGC-27/PTX),采用细胞计数试剂盒(CCK 8)和Transwell小室检测耐药细胞增殖和侵袭能力的变化;采用逆转录实时荧光定量聚合酶链式反应(qRT-PCR)检测耐药细胞中miR-200c表达水平的变化。构建敲低或过表达miR-200c的耐药胃癌细胞系,采用qRT-PCR和蛋白质印迹法检测miR-200c对糖酵解关键酶LDHA、ENO1、HK2表达影响;运用Seahorse能量代谢分析仪检测miR-200c对质子泵出速率、基础糖酵解、补偿糖酵解、细胞外酸化率、基础呼吸和最大呼吸水平的影响。结果AGS细胞和AGS/PTX细胞侵袭率分别为(73.00±5.57)%和(300.33±11.50)%,t=30.81,P<0.001;HGC-27细胞和HGC-27/PTX细胞侵袭率分别为(36.33±3.51)%和(249.00±7.94)%,t=42.44,P<0.001。AGS细胞和AGS/PTX细胞迁移率分别为(140.00±8.00)%和(445.00±9.64)%,t=42.16,P<0.001;HGC-27细胞和HGC-27/PTX细胞迁移率分别为(52.00±5.57)%和(313.00±10.82)%,t=37.16,P<0.001。AGS细胞和AGS/PTX细胞miR200c表达水平分别为1.00±0.00和0.45±0.13,t=7.36,P<0.001;HGC-27细胞和HGC-27/PTX细胞分别为1.00±0.00和0.40±0.03,t=31.03,P<0.001。qRT-PCR和蛋白质印迹实验结果显示,相较于AGS组,AGS/PTX组HK2、ENO1和LDHA表达升高,均P<0.05;相较于HGC-27组,HGC-27/PTX组HK2、ENO1和LDHA表达升高,均P<0.05。相较于AGS/PTX NC组,AGS/PTX OE组HK2、ENO1和LDHA表达降低,均P<0.05;AGS/PTX SI组HK2、ENO1和LDHA表达升高,均P<0.05。相较于HGC-27/PTX NC组,HGC-27/PTX OE组HK2、ENO1和LDHA表达降低;HGC-27/PTX SI组HK2、ENO1和LDHA表达升高,均P<0.05。相较于AGS/PTX NC组,AGS/PTX OE组质子泵出速率、基础糖酵解、补偿糖酵解和细胞外酸化率均降低,基础呼吸和最大呼吸均升高;AGS/PTX SI组质子泵出速率、基础糖酵解、补偿糖酵解和细胞外酸化率均升高,基础呼吸和最大呼吸均降低,均P<0.05。相较于HGC-27/PTX NC组,HGC-27Objective To investigate the role and mechanism of miR-200c in reversing paclitaxel resistance in gastric cancer cells.Methods To construct paclitaxel resistant gastric cancer cell lines(AGS/PTX and HGC-27/PTX),and use cell counting kit(CCK 8)and Transwell chamber to detect changes in the proliferation and invasion ability of drug-resistant cells.Reverse transcription real-time fluorescence quantitative polymerase chain reaction(qRT-PCR)was used to detect changes of miR-200c expression levels in drug-resistant cells.Constructing drug-resistant gastric cancer cell lines with knockdown or overexpression of miR-200c,and detecting the effect of miR-200c on the expression of key glycolytic en-zymes LDHA,ENO1,and HK2 by using qRT PCR and western blotting.The effects of miR-200c on proton pumping rate,basal glycolysis,compensatory glycolysis,extracellular acidification rate,basal respiration,and maximum respira-tion level were detected by using a Seahorse energy metabolism analyzer.Results The invasion rates of AGS cells and AGS/PTX cells were(73.00±5.57)%and(300.33±11.50)%,t=30.81,P<0.001.The invasion rates of HGC-27 cells and HGC-27/PTX cells were(36.33±3.51)%and(249.00±7.94)%,t=42.44,P<0.001.The migration rates of AGS cells and AGS/PTX cells were(140.00±8.00)%and(445.00±9.64)%,t=42.16,P<0.001.The migration rates of HGC-27 cells and HGC-27/PTX cells were(52.00±5.57)%and(313.00±10.82)%,respectively,t=37.16,P<0.001.The expression levels of miR200c in AGS cells and AGS/PTX cells were 1.00±0.00 and 0.45±0.13,t=7.36,P<0.001;1.00±0.00 and 0.40±0.03,t=31.03,P<0.001,respectively.qRT-PCR and western blot experi-ments showed that compared with the AGS group,the expression levels of HK2,ENO1 and LDHA were increased,all P<0.05.Compared with the HGC-27 group,the expression of HK2.ENO1 and LDHA were increased in the HGC-27/PTX group,all P<0.05.Compared with the AGS/PTX NC group,the expression levels of HK2,ENO1 and LDHA in AGS/PTX OE group were decreased,all P<0.05.The expression of HK2,ENO1,and LDHA were increased in t

关 键 词：胃癌 MIR-200C 紫杉醇耐药 糖酵解 能量代谢 

分 类 号：R735.2[医药卫生—肿瘤]