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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 ； |
| 阅读数： | 332 次 |
| 本月下载数： | 7 次 |
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