Chen Jian, Zhang Xing, Wang Yong, Ye Yu, Huang Zhaoquan
School of Basic Medical Sciences, Guilin Medical University, Guilin, China.
Department of Emergency, First Affiliated Hospital of Guangxi Medical University, Nanning, China.
Mol Carcinog. 2016 Mar;55(3):312-9. doi: 10.1002/mc.22282. Epub 2015 Feb 7.
Formononetin is an O-methylated isoflavone that is isolated from the root of Astragalus membranaceus, and it has antitumorigenic effects. Our previous studies found that formononetin triggered growth-inhibitory and apoptotic activities in MCF-7 breast cancer cells. To further investigate the potential effect of formononetin in promoting cell proliferation in estrogen receptor (ER)-positive cells, we used in vivo and in vitro studies to elucidate the possible mechanism. ERα-positive cells (HUVEC, MCF-7) were treated with formononetin. The CCK8 assay, Hoechst 33258, and flow cytometry were used to assess cell proliferation and apoptosis. mRNA levels of ERα, Bcl-2, and miR-375 were quantified using real-time polymerase chain reaction. ERα, p-Akt, and Bcl-2 expression was determined using Western blot. Compared with the control, low formononetin concentrations (2-6 μM) stimulated ERα-positive cell proliferation (HUVEC, MCF-7). The more sensitive HUVEC cells were used to study the relevant signaling pathway. After treatment with formononetin, ERα, miR-375, p-Akt, and Bcl-2 expression was significantly upregulated. The proliferative effect of formononetin was also blocked by a miR-375 inhibitor or raloxifene pretreatment. Additionally, in the in vivo studies, uterine weight in ovariectomized mice treated with formononetin increased significantly, but the weight dramatically decreased with raloxifene or miR-375 inhibitor pretreatment before formononetin. This study demonstrated that formononetin promoted ERα-positive cell proliferation through miR-375 activation and this mechanism is possibly involving in a miR-375 and ERα feedback loop.
芒柄花黄素是一种从黄芪根中分离出的O-甲基化异黄酮，具有抗肿瘤作用。我们之前的研究发现，芒柄花黄素可引发MCF-7乳腺癌细胞的生长抑制和凋亡活性。为了进一步研究芒柄花黄素对雌激素受体（ER）阳性细胞增殖的潜在影响，我们采用体内和体外研究来阐明其可能的机制。用芒柄花黄素处理ERα阳性细胞（人脐静脉内皮细胞、MCF-7）。采用CCK8法、Hoechst 33258染色和流式细胞术评估细胞增殖和凋亡。使用实时聚合酶链反应定量ERα、Bcl-2和miR-375的mRNA水平。使用蛋白质免疫印迹法测定ERα、磷酸化Akt和Bcl-2的表达。与对照组相比，低浓度芒柄花黄素（2-6μM）刺激ERα阳性细胞增殖（人脐静脉内皮细胞、MCF-7）。使用更敏感的人脐静脉内皮细胞来研究相关信号通路。用芒柄花黄素处理后，ERα、miR-375、磷酸化Akt和Bcl-2的表达显著上调。miR-375抑制剂或雷洛昔芬预处理也可阻断芒柄花黄素的增殖作用。此外，在体内研究中，用芒柄花黄素处理的去卵巢小鼠子宫重量显著增加，但在芒柄花黄素处理前用雷洛昔芬或miR-375抑制剂预处理后，子宫重量显著下降。本研究表明，芒柄花黄素通过激活miR-375促进ERα阳性细胞增殖，且该机制可能涉及miR-375与ERα的反馈环。
