miR-181c靶向己糖激酶2抑制癌相关成纤维细胞的糖酵解  

miR-181c inhibits glycolysis by targeting hexokinase 2 in cancer-associated fibroblasts

作　　者：蓝海兵[1] 罗亮[2] 齐协飞[1] 龚园其[1] 陈玉[1] 

机构地区：[1]南昌大学第二附属医院综合ICU,江西南昌330006 [2]中山大学第一附属医院MICU,广东广州510080

出　　处：《南方医科大学学报》2015年第11期1619-1623,共5页Journal of Southern Medical University

摘　　要：目的探讨mi R-181c在癌相关成纤维细胞(CAFs)糖酵解中的作用和机制。方法用组织贴壁法分离原代肺腺癌病人肺组织CAF及肺正常成纤维细胞(NFs)。用LipofectamineTM2000转染mi R-181c mimics,mi R-181c inhibitor,si RNA-HK2和HK2-vecto等;Q-PCR检测mi R-181家族表达水平;Western blotting检测己糖激酶2(HK2)蛋白表达;2-NBDG法检测细胞葡萄糖摄取率;DRY-CHEM FCD3500仪器检测细胞上清中乳酸生成;双荧光素酶报告基因系统检测HK2 m RNA表达。结果在细胞形态上CAFs与NFs无明显区别。与NFs组相比,CAFs组葡萄糖摄取、乳酸生成和HK2蛋白表达明显增加,而mi R-181家族表达明显减少(P<0.05)。在NFs转染inhibitor-181c后,其HK2蛋白表达、葡萄糖摄取和乳酸生成明显增加(P<0.05);相反,CAFs转染了mimics-181c后,其HK2蛋白表达、葡萄糖摄取和乳酸生成明显减少(P<0.05)。并且在敲低HK2细胞的CAFs中,mimics-181c不能减少葡萄糖摄取和乳酸生成;而mimics-181c可以减少过表达HK2对NFs葡萄糖摄取和乳酸生成的增强作用。结论 mir-181c可以通过抑制HK2的蛋白表达来抑制CAFs的糖酵解。Objective To investigate the role of mi R-181 c in glycolysis of cancer-associated fibroblasts（CAFs） and explore the mechanism. Methods Human lung CAFs and normal fibroblasts（NFs）, isolated from fresh human lung adenocarcinoma tissue specimens by primary culture of tissue explants, were transfected with a mi R-181 c mimics, a mi R-181 c inhibitor, a si RNA si RNA-HK2 or the vector HK2-vector via LipofectamineTM2000. Quantitative real-time PCR was used to analyze the changes in mi R-125 b expression in the transfected cells; hexokinase-2（HK2） protein expression in the cells was detected using Western blotting, and the cellular glucose uptake was assessed with 2-NBDG. Lactate production in the cells was examined and expression of HK2 m RNA was detected with dual luciferase reporter gene assay. Results No obvious difference was found in the cell morphology between CAFs and NFs. Compared with the NFs, the CAFs showed obviously increased glucose uptake,lactate production and HK2 protein expression with decreased expressions of the mi R-181 family（P〈0.05）. Transfection with the mi R-181 inhibito- rsignificantly increased glucose uptake, lactate production and HK2 protein expression in the NFs. In CAFs, transfection with the mi R-181 mimics caused significantly lowered glucose uptake, lactate production and HK2 protein expression of. Knockdown of endogenous HK2 by si RNA abolished mi R-181 mimics-mediated decrease of glucose uptake and lactate production in CAFs, while transfection with mi R-181 mimics suppressed HK2 overexpression-induced enhancement of glucose uptake and lactate production in NFs. Conclusion Transfection with mi R-181 mimics can suppress glycolysis in CAFs by inhibiting HK2 expression.

关 键 词：癌相关成纤维细胞 miR-181 己糖激酶2 糖酵解 

分 类 号：R73-36[医药卫生—肿瘤]