ERK途径在胰岛素促进子宫内膜癌细胞增殖中的作用

Mitogenic Effect Induced by Insulin in Endometrial Cancer Cells are Via Ras/ERK Signaling Pathway

背景与目的观察胰岛素对子宫内膜癌细胞ERK信号通路的活化,并探讨ERK信号通路对CyclinD1 mRNA、蛋白水平的影响及该通路在细胞增殖调节中的作用。方法将无血清饥饿的子宫内膜癌Ishikawa3-H^-12细胞分为空白对照组、10-6mol/L胰岛素单独刺激组以及不同剂量MEK抑制剂PD98059预处理后再用胰岛素刺激组。Western blot检测各组ERK磷酸化（p-ERK）及CyclinD1蛋白水平,RT-PCR检测CyclinD1 mRNA水平,MTT试验观察细胞增殖情况。结果胰岛素可引起子宫内膜癌细胞ERK活化,刺激30min后p-ERK/ERK比值显著高于空白对照组（65.41%vs22.45%,P〈0.001）,胰岛素可促进子宫内膜癌细胞CyclinD1mRNA转录和蛋白表达,分别刺激12h、24h后CyclinD1mRNA转录和蛋白表达水平显著高于空白对照组（分别为84.62%vs18.55%;74.04%vs32.91%,均P〈0.001）,PD98059以浓度依赖方式抑制胰岛素引起的ERK磷酸化、mRNA转录和蛋白表达水平。MTT试验显示,在药物处理24h,48h和72h3个时间点,不同组别570nm吸光度值（OD570）均有显著差异（F=204.160;33.850;90.764,均P〈0.001）。胰岛素组OD570值均高于同时间点的空白对照组（均P〈0.001）,PD98059可抑制胰岛素的增殖促进作用,且具有浓度依赖性。结论胰岛素可以经由ERK途径促进CyclinD1 mRNA转录和蛋白表达,并进一步促进细胞增殖。

Background and objective To explore the effects of insulin on activation of extracellular signal regulated kinase （ERK） and Cyclin DlmRNA and protein levels. Study the role of ERK signal pathway on cell proliferation. Methods Ishikawa 3-H-1 2 human endometrial cancer cells were serum-starved and then stimulated by insulin with or without PD98059-a specific inhibitor of MEK. The phosphorylation level of ERK （p-ERK） and Cyclin D 1 expression were detected with Western blots. Cyclin D lmRNA were observed by RT-PCR. Cellular proliferation was determined with the MTF assay. Results Stimulation of the Ishikawa cells with 10.6 mol/L insulin resulted in the activation of ERK. After 30 min, the p-ERK/ERK ratio was significantly higher than control （65.41% vs 22.45%, P〈0.001）. Insulin increased Cyclin D lmRNA and protein levels. After 12 h and 24 h respectively, the levels of Cyclin DlmRNA and protein were significantly higher than control （84.62% vs 18.55%; 74.04% vs 32.91%, both P〈0.001 ）. Insulin induced ERK activation and Cyclin D 1 mRNA transcription and protein expression were inhibited by PD98059 in concentration -dependent manners. In M＇/T assay, OD570 of different groups in 24 h, 48 h and 72 h were measured. At each time point, there was significant difference among six groups （F=204.160; 33.850; 90.764, both P〈0.001）. After insulin treatment, the OD570 in 24 h, 48 h and 72 h was all higher than that in control group （all P〈0.001）. PD98059 inhibited the mitogenic effect induced by insulin in a dose-dependent manner. Conclusion Insuin induces Cyclin DImRNA transcription and protein expression and promotes Cellular proliferation via ERK signal pathway.