基于Calpain-ERK信号通路研究Calpeptin对雌激素诱导人乳腺上皮细胞MCF-10A转化及干性标志物表达的影响  

Study on the Effects of Calpeptin on Estrogen-induced Transformation and Stemness Markers Expression of Mammary Epithelial Cells MCF-10A Based on Calpain-ERK Signaling Pathway

作　　者：张艳 王旭东 金爱 何艳 詹云惠 沈敬堃 董宇华 宛蕾 ZHANG Yan;WANG Xudong;JIN Ai;HE Yan;ZHAN Yunhui;SHEN Jingkun;DONG Yuhua;WAN Lei(Dept.of Physiology,School of Basic Medical Sciences,Guizhou Medical University,Guiyang 550025,China;Dept.of Pharmacology,School of Basic Medical Sciences,Guizhou Medical University,Guiyang 550025,China;Dept.of Ultrasonography,Guiyang First People’s Hospital,Guiyang 550002,China)

机构地区：[1]贵州医科大学基础医学院生理学教研室,贵阳550025 [2]贵州医科大学基础医学院药理学教研室,贵阳550025 [3]贵阳市第一人民医院超声科,贵阳550002

出　　处：《中国药房》2020年第13期1549-1556,共8页China Pharmacy

基　　金：国家自然科学基金资助项目(No.31660345,No.31360252)。

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+1μmol/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)m RNA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(10μmol/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 m RNA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,其机制可能与抑制Calpain-ERK信号通路�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+1μmol/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(10μmol/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

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 

分 类 号：R737.9[医药卫生—肿瘤] R34[医药卫生—临床医学]