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

发布时间：2018-01-30 00:26

本文关键词： 乳腺肿瘤瘦素果糖-二磷酸酶细胞增殖细胞凋亡　出处：《肿瘤》2017年06期 　论文类型：期刊论文

【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。
[Abstract]:Objective: to investigate the effects of leptin on proliferation and apoptosis of human breast cancer BT-474 and MDA-MB-231 cells. Methods: human breast cancer BT-474 and MDA-MB-231 cells were treated with leptin 200 ng / mL. The proliferation and apoptosis of BT-474 and MDA-MB-231 cells were detected by CCK-8 assay and FCM assay respectively. Detection of proliferation-associated proteins c-Myc and cyclin by Western blotting. The expression of D1 and apoptosis-related protein Bcl-2 and Bax were detected by real-time fluorescence quantitative PCR and Western blotting respectively. 200 ng / mL). Fructose-1 fructose-1 bisphosphatase 1 in BT-474 and MDAMB-231 cells. The expression level of FBP1)mRNA and protein was changed. Then, the liposome transfection method was used. The recombinant plasmid carrying FBP 1 gene was transfected into BT-474 and MDA-MB-231 cells, and then treated with leptin 200 ng / mL. The expression of FBP1 mRNA and its protein was detected by real-time fluorescence quantitative PCR and Western blotting. In order to verify the effect of overexpression of FBP 1 gene, the proliferative ability of BT-474 and MDA-MB-231 cells was detected again by CCK-8 assay. The expression of proliferation-associated protein c-Myc and cyclin D1 was detected by Western blotting and apoptosis rate was detected by FCM. The expression levels of Bcl-2 and Bax of apoptosis-related proteins were detected by Western blotting. Results: after treated with leptin 200 ng / mL. The proliferative ability of BT-474 and MDA-MB-231 cells increased significantly (P < 0.01). The expression of proliferation-associated protein c-Myc and cyclin D1 increased significantly (P < 0.01). At the same time, the percentage of apoptosis decreased significantly (P < 0.01). The expression of apoptosis-related protein Bcl-2 increased significantly (P < 0.01). The expression of FBP1 mRNA and protein were significantly decreased by Bax expression in both 0. 01 and 0. 2 cells compared with those before leptin treatment (P < 0. 05). The BT-474 and MDA-MB-231 cells overexpressed by FBP1 were treated with leptin. The expression of FBP1 mRNA and protein increased significantly (P < 0.01), and inhibited the proliferation of BT-474 and MDA-MB-231 cells. Conclusion: leptin can promote the proliferation of human breast cancer BT-474 and MDA-MB-231 cells and inhibit the apoptosis of two cell lines. The mechanism may be related to the down-regulation of FBP 1 gene expression.
【作者单位】：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;
【基金】：国家自然科学基金资助项目(编号:81272544)~~
【分类号】：R737.9
【正文快照】： 乳腺癌是中国女性发病率最高的癌症,也是中国女性恶性肿瘤的第6位死亡原因[1]。瘦素(leptin)是一类由肥胖基因编码、由脂肪细胞合成分泌的多肽类激素。瘦素除了能调节机体能量平衡、维持正常生殖周期和介导免疫应答之外,还可特异性地与其受体结合,通过激活下游的信号转导蛋白,

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