IGFBP7对人乳腺癌MCF-7细胞增殖的影响及其机制

Effect of IGFBP7 on proliferation of human breast cancer cell line MCF-7

目的:探究胰岛素样生长因子结合蛋白7(IGFBP7)对人乳腺癌MCF-7细胞增殖的影响及其分子生物学机制。方法:将质粒pCMV6-IGFBP7转染MCF-7细胞,构建稳定表达IGFBP7的MCF-7细胞系;采用Western blotting检测IGFBP7在MCF-7细胞稳定转染子的表达;采用软琼脂培养克隆形成实验检测IGFBP7对MCF-7细胞克隆形成能力的影响;采用流式细胞术检测IGFBP7对MCF-7细胞周期的影响;采用Western blotting检测IGFBP7对MCF-7细胞细胞外信号调节激酶1/2(ERK1/2)、p-ERK1/2、细胞周期素D1(cyclin D1)、细胞周期素依赖性激酶4(CDK4)、cyclin E、CDK2、p21CIP1/WAF1、p27KIP1、p53、视网膜母细胞瘤蛋白(Rb)和p-Rb蛋白含量的影响。结果:(1)只有稳定转染质粒pCMV6-IGFBP7的MCF-7细胞表达IGFBP7。(2)IGFBP7能够显著降低MCF-7细胞的克隆形成率(P<0.01),阻止细胞从G1期进入S期,使其停滞于G1期(P<0.01)。(3)IGFBP7能够显著抑制ERK1/2的磷酸化(P<0.01)。(4)IGFBP7能够下调cyclin D1和cyclin E蛋白表达(P<0.01),上调p27KIP1、p21CIP1/WAF1和p53蛋白表达(P<0.01),抑制Rb的磷酸化(P<0.01)。(5)MEK1/2阻断剂PD98059可部分模拟IGFBP7的肿瘤抑制效应。结论:(1)IGFBP7可通过下调cyclin D1和cyclin E蛋白表达,上调p27KIP1、p21CIP1/WAF1和p53蛋白表达,以及抑制Rb磷酸化发挥抗肿瘤作用;(2)IGFBP7对cyclin D1和p27KIP1的调节可能与其抑制ERK1/2信号通路有关。

AIM： To investigate the mechanism that insulin -like growth factor binding protein 7 （IGFBP7） inhibits proliferation of human breast cancer cell line MCF - 7. METHODS： Plasmid pCMV6 - IGFBP7 or empty plasmid was transfected into MCF -7 cells. The expression of IGFBP7 in MCF -7 cells after transfection was detected by Western blotting. The effects of IGFBP7 on the colony - forming efficiency and the cell cycle were studied by soft agar colony formation assay and flow cytometry,respectively. The effects of IGFBP7 on the expression of ERK1/2, p - ERK1/2, cyclin D1, CDK4, cyclin E, CDK2, p21CIPI/WAF1 p27KIP1, p53, Rb and p -Rb in MCF -7 cells were detected by Western blotting. RESULTS： Only the transfectant of pCMV6 - IGFBP7 expressed IGFBP7. IGFBP7 remarkably reduced colony - forming efficiency （P 〈0.01 ） and G0/G1 arrest （P 〈0.01 ）, inhibited phosphorylation of ERK1/2 （P 〈0.01 ）, down - regulated cyclin D1 and cyclin E （P 〈0.01 ）, up- regulated p27rat1, p21ctPt/wAF1 and IX53 （P 〈0.01 ）, and inhibited phosphorylation of Rb （P 〈 0.01 ） in MCF - 7 cells. PD98059, an inhibitor of MEK1 and MEK2, imitated part of the tumor - suppressing activity of IGFBP7. CONCLUSION： IGFBP7 inhibits the proliferation of human breast cancer cell line MCF- 7 by down -regulating cyclin D1 and cyclin E, up -regulating p27KIP1 , p21CIP1/WAF1 and p53 and inhibiting phosphorylation of Rb. ERK1/2 signaling pathway might be involved in the regulation of cyclin D1 and p27KIP1 by IGFBP7.