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
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篇名: 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
TITLE: 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
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(50nmol/L)、E2转化+Calpeptin组(50nmol/LE2+1μmol/LCalpeptin),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、48h),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Westernblotting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、48h)、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-ERKmRNA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、48h)、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、ERKmRNA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信号通路的激活有关。
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 E 2 treatment. Cells were divided into control group (0.1%DMSO), E2-transformed group (50 nmol/L),E2-transformed+Calpeptin group (50 nmol/L E 2+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 CD 44,Nanog,OCT4 ,ERK and p-ERK were detected by Western blotting assay. Another E 2-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 CD 44,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 E 2 transformed group were increased significantly (P<0.01),and the number of sphere-forming was increased significantly(P<0.01);mRNA expression levels of CD 44,Nanog,OCT4,ERK and protein expression levels of CD 44,Nanog, OCT4 and p-ERK in cells were increased significantly (P<0.01). Compared with E 2-transformed group ,proliferation rate (24,48 h)and clone formation rate of E 2-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 CD 44,Nanog,OCT4 ,ERK and protein expression levels of CD 44,Nanog,OCT4,p-ERK in cells were decreased significantly (P<0.05 or P<0.01). After treated with ERK inhibitor U 0126,clone formation rate of E 2-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.
期刊: 2020年第31卷第13期
作者: 张艳,王旭东,金爱,何艳,詹云惠,沈敬堃,董宇华,宛蕾
AUTHORS: ZHANG Yan,WANG Xudong ,JIN Ai,HE Yan,ZHAN Yunhui ,SHEN Jingkun ,DONG Yuhua ,WAN Lei
关键字: 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
KEYWORDS: Calpeptin;Estradiol;Human mammary epithelial cells MCF- 10A;Cell transformation ;Stemness marker ;
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