张艳 王旭东 金爱 何艳 詹云惠 沈敬堃 董宇华 宛蕾

摘 要 目的：研究钙激活中性蛋白酶（Calpain）抑制剂Calpeptin对雌二醇（E2）诱导人乳腺上皮细胞MCF-10A转化及干性标志物表达的影响，并探讨其作用机制。方法：以人乳腺上皮细胞MCF-10A为研究对象，采用E2诱导制备转化细胞模型。将细胞分为对照组[0.1%二甲基亚砜（DMSO）]、E2转化组（50 nmol/L）、E2转化+Calpeptin组（50 nmol/L E2+1 μmol/L Calpeptin），以相应含药培养基连续培养15代。然后采用MTT法检测细胞的增殖率（24、48 h），采用平板克隆试验检测细胞的克隆形成率，采用悬浮成球试验检测细胞的成球数;采用实时荧光定量-聚合酶链式反应法检测细胞中干性标志物（CD44、Nanog、OCT4）和细胞外信号调节激酶（ERK）mRNA表达水平，并采用Western blotting法检测细胞中CD44、Nanog、OCT4、ERK和磷酸化ERK（p-ERK）蛋白表达水平。另取E2转化细胞分为对照组（0.1%DMSO）和U0126（ERK抑制剂）组（10 μmol/L），按上述方法测定细胞的克隆形成率、成球数以及细胞中CD44、Nanog、OCT4、p-ERK蛋白表达水平，以验证ERK表达抑制与转化细胞生物学行为及干性标志物表达之间的关系。结果：与对照组比较，E2转化组细胞的增殖率（24、48 h）、克隆形成率均显著升高（P<0.01），细胞成球数均显著增加（P<0.01），细胞中CD44、Nanog、OCT4、p-ERK mRNA表达水平及CD44、Nanog、OCT4、p-ERK蛋白表达水平均显著升高（P<0.01）。与E2转化组比较，E2转化+Calpeptin组细胞的增殖率（24、48 h）、克隆形成率均显著降低（P<0.01），细胞成球数显著减少（P<0.05），细胞中CD44、Nanog、OCT4、ERK mRNA表达水平及CD44、Nanog、OCT4、p-ERK蛋白表达水平均显著降低（P<0.05或P<0.01）。加入ERK抑制剂U0126后，E2转化细胞的克隆形成率、成球数以及细胞中p-ERK、CD44、Nanog、OCT4蛋白表达水平均显著增加或升高（P<0.05或P<0.01）。结论：Calpeptin可抑制E2诱导的人乳腺上皮细胞MCF-10A转化及干性标志物表达，其機制可能与抑制Calpain-ERK信号通路的激活有关。

关键词 Calpeptin;雌二醇;人乳腺上皮细胞MCF-10A;细胞转化;干性标志物;细胞外信号调节激酶;机制

ABSTRACT   OBJECTIVE： To study the effects of Calpeptin inhibitor Calpeptin on the transformation and stemness markers expression induced by estradiol （E2）， and to investigate its mechanism. METHODS： Taking human mammary epithelial cells MCF-10A as research object， transformed cells were induced by E2 treatment. Cells were divided into control group（0.1%DMSO）， E2-transformed group （50 nmol/L）， E2-transformed+Calpeptin group （50 nmol/L E2+1 μmol/L Calpeptin）， then continuously treated with corresponding drug-containing culture medium for 15 generations. Then， MTT assay was used to determine the proliferation rate of cells （24，48 h）; plate colony test was used to detect the Clone formation rate of cells; the number of sphere-forming cells was measured by suspension spheroidization test;mRNA expressions of stemness marker （CD44， Nanog， OCT4） and extracellular sigal-regulated kinase （ERK） were detected by RT-qPCR，and protein expressions of CD44， Nanog， OCT4 ， ERK and p-ERK were detected by Western blotting assay. Another E2-transformed cells were divided into control group （0.1%DMSO） and U0126 （ERK inhibitor） group （10 μmol/L）. Clone formation rate， the number of sphere-forming， protein expressions of CD44， Nanog， OCT4，ERK and p-ERK were determined with above methods， and to validate the relationship of ERK inhibition with transformed cell behavior and the expression of stemness markers. RESULTS：Compared with control group， proliferation rate and clone formation rate of E2 transformed group were increased significantly （P<0.01）， and the number of sphere-forming was increased significantly （P<0.01）; mRNA expression levels of CD44， Nanog， OCT4，ERK and protein expression levels of CD44， Nanog， OCT4 and p-ERK in cells were increased significantly （P<0.01）. Compared with E2-transformed group， proliferation rate （24， 48 h） and clone formation rate of E2-transformed+Calpeptin group were decreased significantly （P<0.01）， and the number of sphere-forming was decreased significantly （P<0.05）; mRNA expression levels of CD44， Nanog， OCT4 ， ERK and protein expression levels of CD44， Nanog， OCT4， p-ERK in cells were decreased significantly （P<0.05 or P<0.01）. After treated with ERK inhibitor U0126， clone formation rate of E2-transformed cells， the number of sphere-forming， protein expression levels of CD44， Nanog， OCT4 and p-ERK were increased significantly （P<0.05 or P<0.01）. CONCLUSIONS： Calpeptin can inhibit the transformation and the expression of stemness markers of human mammary epithelial cells MCF-10A， and the mechanism of it may be associated with inhibiting the activation of Calpain-ERK signaling pathway.

KEYWORDS   Calpeptin; Estradiol; Human mammary epithelial cells MCF-10A; Cell transformation; Stemness marker; Extracellular sigal-regulated kinase; Mechanism

雌二醇（E2）是诱导乳腺癌发生的主要风险因素[1]，其可通过激活雌激素受体（ER）或代谢产生有毒代谢产物，从而诱导乳腺上皮细胞转化以及癌变发生[2-3]。相关研究显示，乳腺肿瘤干细胞（BCSCs）在乳腺癌的发生、维持、转移、治疗抵抗和復发中起着重要作用，而E2与BCSCs的产生密切相关[4]。人乳腺上皮细胞MCF-10A是一种非致瘤性乳腺上皮细胞，正常情况下其呈不规则多边形贴壁生长，且增殖缓慢;但经E2长期诱导后，MCF-10A细胞将失去上皮细胞相关特性，胞体变大，呈长梭形贴壁生长，增殖能力增强，且具有一定的间质特征[5]。……