瘦素通过下调FBP1基因表达促进人乳腺癌BT-474和MDA-MB-231细胞增殖并抑制细胞凋亡  

Leptin promotes proliferation and inhibits apoptosis of human breast cancer BT-474 and MDA-MB-231 cells by down-regulating the expression of FBP1 gene

作　　者：刘一锋[1] 蒋玉林[1] 张志乾[1] 杨俊鸿[1] 靳倩妮 叶相森 陈婷梅[1] 

机构地区：[1]重庆医科大学检验医学院临床检验诊断学教育部重点实验室,重庆400016

出　　处：《肿瘤》2017年第6期556-567,共12页Tumor

基　　金：国家自然科学基金资助项目(编号:81272544)~~

摘　　要：目的:探讨瘦素(leptin)对人乳腺癌BT-474和MDA-MB-231细胞增殖和凋亡的影响,及其可能的分子作用机制。方法:应用瘦素(200 ng/mL)处理人乳腺癌BT-474和MDA-MB-231细胞后,分别采用CCK-8法和FCM法检测BT-474和MDA-MB-231细胞的增殖及凋亡能力;蛋白质印迹法检测增殖相关蛋白c-Myc和cyclin D1以及凋亡相关蛋白Bcl-2和Bax表达的变化。采用实时荧光定量PCR和蛋白质印迹法分别检测经瘦素(200 ng/mL)处理后,BT-474和MDAMB-231细胞内果糖-1,6-二磷酸酶1(fructose-1,6-bisphosphatase 1,FBP1)mRNA和蛋白表达水平的变化。然后,采用脂质体转染法,将携带有FBP 1基因的重组质粒分别转染至BT-474和MDA-MB-231细胞中,随后行瘦素(200 ng/mL)处理,再应用实时荧光定量PCR和蛋白质印迹法检测FBP1 mRNA及其蛋白的表达水平,以验证FBP 1基因过表达的效果。采用CCK-8法再次检测BT-474和MDA-MB-231细胞的增殖能力的变化,蛋白质印迹法检测增殖相关蛋白c-Myc和cyclin D1表达水平的变化,FCM法检测细胞凋亡率的变化,并采用蛋白质印迹法检测凋亡相关蛋白Bcl-2和Bax表达水平的变化。结果:经瘦素(200 ng/mL)处理后,BT-474和MDA-MB-231细胞的增殖能力明显增强(P值均<0.01);增殖相关蛋白c-Myc和cyclin D1的表达水平明显升高(P值均<0.01);同时,细胞凋亡率明显减少(P值均<0.01);凋亡相关蛋白Bcl-2表达水平明显升高(P值均<0.01),Bax表达水平明显降低(P值均<0.01)。2株细胞中FBP1 mRNA及蛋白的表达水平均较未用瘦素处理前明显下调(P值均<0.05)。FBP1过表达的BT-474和MDA-MB-231细胞经瘦素处理后,FBP1 mRNA及蛋白的表达水平均明显上调(P值均<0.01),并抑制BT-474和MDA-MB-231细胞的增殖能力,促进细胞的抗凋亡能力(P值均<0.01)。结论:瘦素可促进人乳腺癌BT-474和MDA-MB-231细胞的增殖并抑制2株细胞的凋亡,其机制可能与下调FBP 1基因表达有关。Objective： To investigate the effects of leptin on the proliferation and apoptosis of human breast cancer BT-474 and MDA-MB-231 cells, and to explore its underlying molecular mechanism.Methods： After treatment with 200 ng/mL leptin, the proliferation and apoptosis of BT-474 and MDA-MB-231 cells were measured by CCK-8 assay and FCM, respectively; the expression levels of proliferation-and apoptosis-associated proteins c-myc, cyclin D1, Bcl-2 and Bax were determined by Western blotting; while the levels of fructose-1, 6-bisphosphatase 1 （FBP1） mRNA and protein were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. After the recombinant plasmids carrying FBP1 gene were transfected by liposome into BT-474 and MDA-MB-231 cells which were treated with 200 ng/mL leptin, the expression levels of FBP1 mRNA and protein were detected by real-time fluorescent quantitative PCR and Western blotting to verify the over-expression of FBP1 gene. Then, the proliferation abilities of BT-474 and MDA-MB-231 cells were measured by CCK-8 assay, the expression levels of proliferation-associated proteins c-myc and cyclin D1 were determined by Western blotting, the apoptosis rates of the two cell lines were detected by FCM, and the expression levels of apoptosis-associated proteins Bcl-2 and Bax were determined by Western blotting.Results： After treatment with 200 ng/mL leptin, the proliferation abilities of human breast cancer BT-474 and MDA-MB-231 cells were significantly increased （both P 〈 0.01）; the expressions of proliferation-associated proteins c-myc and cyclin D1 were significantly up-regulated （both P 〈 0.01）; while the apoptosis rates were apparently reduced （both P 〈 0.01）; the expression of apoptosis-associated protein Bcl-2 was apparently up-regulated （P 〈 0.01）, but the expression of Bax was apparently down-regulated （P 〈 0.01）; the mRNA and protein levels of FBP1 in BT-474 and MDA-MB-231 cells were obviously down-regulated （both P 〈 0.05）. Ho

关 键 词：乳腺肿瘤 瘦素 果糖-二磷酸酶 细胞增殖 细胞凋亡 

分 类 号：R737.9[医药卫生—肿瘤]