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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 effect of leptin on the proliferation and apoptosis of human breast cancer cell lines BT-474 and MDA-MB-231 and its possible molecular mechanism. Methods: Human breast cancer cell lines BT-474 and MDA-MB-231 were treated with 200 ng / mL leptin and the proliferation of BT-474 and MDA-MB-231 cells were detected by CCK- The apoptotic ability was detected by Western blotting. The expressions of c-Myc, cyclin D1 and Bcl-2 and Bax were detected by Western blotting. Real-time fluorescence quantitative PCR and Western blotting were used to detect the expression of fructose-1,6-bisphosphatase in BT-474 and MDAMB-231 cells treated with 200 ng / mL leptin 1, FBP1) mRNA and protein expression levels. Then, the recombinant plasmids carrying FBP1 gene were transfected into BT-474 and MDA-MB-231 cells respectively by lipofection method, then treated with leptin (200 ng / mL) Fluorescent quantitative PCR and Western blotting were used to detect the expression of FBP1 mRNA and protein in order to verify the effect of FBP1 gene overexpression. The proliferation of BT-474 and MDA-MB-231 cells were detected by CCK-8 method. The expression of c-Myc and cyclin D1 were detected by Western blotting. The changes of apoptosis rate were detected by FCM , And Western blotting was used to detect the expression of Bcl-2 and Bax. Results: The proliferation of BT-474 and MDA-MB-231 cells was significantly enhanced by leptin (200 ng / mL) (P <0.01), and the expression levels of c-Myc and cyclin D1 (P <0.01). At the same time, the apoptosis rate was significantly decreased (P <0.01), the expression of Bcl-2 was significantly increased (P <0.01), and the expression of Bax was significantly higher Decrease (all P <0.01). The FBP1 mRNA and protein expression levels in two cell lines were significantly lower than those before leptin treatment (all P <0.05). FBP1 overexpression in BT-474 and MDA-MB-231 cells after leptin treatment, FBP1 mRNA and protein expression levels were significantly increased (P all <0.01), and inhibit BT-474 and MDA-MB-231 cells Proliferative ability and anti-apoptotic ability of cells (all P <0.01). Conclusion: Leptin can promote the proliferation of human breast cancer BT-474 and MDA-MB-231 cells and inhibit the apoptosis of the two cell lines, which may be related to the down-regulation of FBP 1 gene expression.