MAPKs通路在Leptin促人乳腺癌细胞系增殖中的机制研究

Role of MAPKs signal transduction system in the proliferation of mammary carcinoma cell line MCF-7 induced by Leptin

目的:探讨MAPKs信号转导通路在瘦素(Leptin)促人乳腺癌细胞MCF-7增殖中的作用机制。方法:用MTT比色法观察不同浓度瘦素促MCF-7细胞的增殖效应,以及用JNK、ERK特异性抑制剂(SP600125,PD98059)阻断MAPKs信号通路对瘦素促MCF-7细胞增殖效应的影响;Westem blot检测瘦素干预不同时间对p-JNK、JNK、p-ERK、ERK蛋白表达水平的影响;RT-PCR检测在瘦素作用下以及MAPKs信号通路阻断情况下对MCF-7细胞表达瘦素受体(Ob-R)mRNA水平的影响。结果:50 ng/ml瘦素促人乳腺癌细胞MCF-7增殖效应最强,该浓度瘦素可使MAPKs信号通路中的靶蛋白发生磷酸化激活;50 ng/ml瘦素作用于MCF-7细胞30 s后p-ERK蛋白表达即显著增加,3 min后p-JNK蛋白表达亦显著增加;分别用SP600125和PD98059阻断JNK、ERK信号通路可显著抑制瘦素促MCF-7细胞的增殖效应.亦可抑制瘦素作用下MCF-7细胞Ob-R mRNA表达水平的增加。结论:瘦素促人乳腺癌细胞MCF-7增殖效应可能与激活MAPKs信号通路有关,亦调控MCF-7细胞中瘦素受体的表达,在肿瘤细胞周围形成瘦素自分泌环,促进乳腺癌的发生发展。

Objective：To explore the role of MAPKs signal transduction system in the proliferation of human mammary carcinoma cell line MCF-7 induced by Leptin. Methods：Human mammary carcinoma cell line MCF-7 was treated with different concentrations of Leptin. The proliferation of MCF-7 ceils was measured by MTr assay. The expression of Ob-R mRNA was detected by RT-PCR with or without MAPKs signals blocked by their each inhibitors,SP600125,the inhibitor of JNK,and PD98059,the inhibitor of ERK. The expression of JNK, p-JNK, ERK, p-ERK was detected at different time treated with leptin by Western blot. Results：Leptin increased the proliferation of MCF-7 cells in a concentration-dependent manner. Western blotting vesuhs implicated that 50 ng/ml Lep- tin induced the activation of JNK and ERK. The expression of p-JNK was detected with 3 rain after Leptin treated and at the same time rapidly reached its peak. While the ERK was activated earlier than the JNK,at the 304 second after Leptin treated. The protein content of total JNK and ERK had no significant changes. Then the proliferation activated by leptin after MAPKs signaling pathway blocked by their inhibitors was reevaluated. The results showed that after inhibiting the activities of JNK and ERK,the proliferation induced by Leptin was also attenuated. Otherwise, the level of Leptin receptor expressing on the surface of MCF-7 cells was examined,which formed an autocrine loop and facilitated the effect of Leptin. 100 ng/ml Leptin increased the Oh-R mRNA level,while blocking the MAPKs signaling pathway decreased the Ob-R mRNA level. Conclusion：h demonstrated that leptin can stimulate the proliferation of MCF-7 cells in vitro. MAPKs signal transduction system involved in the proliferation, which also regulated the expression of Ob-R,forming an autocrine loop at the local site of tumor and amplifying the leptin signaling pathway.