杨琼,王婧雅,王璐,周培富,何可群,严敏,毛大华,陈腾祥,张金娟
YANG Qiong,WANG Jingya,WANG Lu,ZHOU Peifu,HE Kequn,YAN Min,MAO Dahua,CHEN Tengxiang,ZHANG Jinjuan

• 1:贵州民族大学民族医药学院
• 2:贵州医科大学基础医学院
• 3:贵州医科大学附属乌当医院

摘要(Abstract)：

目的 研究雌二醇通过calpain 2/integrin β4促进人表皮生长因子受体(Her2)阳性的乳腺癌SK-Br3细胞迁移和浸润的相关机制。方法 常规培养乳腺癌SK-Br3细胞，calpain 2的特异性抑制剂或雌二醇(E2)处理细胞，并对汇合度为60%(低密度)和90%(高密度)的细胞进行培养；通过细胞划痕实验检测SK-Br3细胞的迁移能力，Transwell-Matrigel实验检测SK-Br3细胞的浸润能力，免疫细胞化学技术和激光共聚焦扫描显微镜观察integrin β4和Her2的表达与共定位，免疫共沉淀技术检测integrin β4和Her2的相互作用，Western blot技术检测相关蛋白或蛋白片段的表达及蛋白磷酸化水平。结果 划痕和浸润实验显示，相对于对照组，Calpain2抑制剂处理的SK-Br3细胞迁移和浸润能力减弱(P<0.05);免疫细胞化学成像显示，Her2和integrin β4的共定位主要在细胞膜上，在胞浆也有少量共定位，用calpain inhibitorⅣ处理后，Her2和integrin β4的表达减少，共定位也明显减少；免疫共沉淀实验发现，在Her2免疫沉淀物中可检测到integrin β4,在integrin β4免疫沉淀物中可检测到Her2;给予calpain inhibitorⅣ处理，可以减少Her2和integrin β4之间的免疫共沉淀；Western blot检测发现，高密度培养乳腺癌SK-Br3细胞中的integrin β4比低密度培养的水解程度高；在高密度培养条件下，E2可以促进这些integrin β4水解片段的产生，上调calpain 2的表达，也伴随着integrin β4水解程度的增加，同时使Src的Y418位点的磷酸化水平增高。结论 高密度培养条件下，E2可通过上调和激活calpain2,促进integrin β4的有限水解，促进integrin β4和Her2的相互作用，磷酸化激活Src,促进SK-Br3细胞的迁移和浸润。
Objective To study the mechanism by which estradiol promotes the migration and invasion of human epidermal growth factor receptor 2(Her2)-positive SK-Br3 breast cancer cells through calpain 2/integrin β4.Methods SK-Br3 breast cancer cells were cultivated conventionally, or treated by calpain 2 specific inhibitors or estradiol(E2), and cultivated with 60%(low density) and 90%(high density) confluence. The migration ability of SK-Br3 cells was tested by wound healing assay, and the invasion ability was tested by Transwell-Matrigel assay; the expression and co-localization of integrin β4 and Her2 were detected by immunocytochemical technique and laser confocal scanning microscope; the interaction between integrin β4 and Her2 was detected by co-immunoprecipitation assay; Western blot technology was adopted to detect the expression and phosphorylation level of related proteins or protein fragments. Results Wound healing assay and invasion experiments showed that, compared with control group, the migration and invasion ability of SK-Br3 cells treated with Calpain2 inhibitors was weakened(P<0.05); immunocytochemical imaging showed that the major colocalization of Her2 and integrin β4 was on the cell membrane and the minor in the cytoplasm. After treatment with calpain inhibitor Ⅳ, the expression of Her2 and integrin β4 reduced and the colocalization also decreased significantly. Immunoprecipitation experiments showed that integrin β4 could be detected in Her2 immunoprecipitate, and Her2 could also be detected in the integrin β4 immunoprecipitate, both of which could be reduced after treatment with calpain inhibitor Ⅳ. The result of Western blot found that hydrolysis degree of integrin β4 in breast cancer SK-Br3 cells cultured in high density was higher than that cultured in low density. Under high-density culture conditions, E2 could promote the formation of these integrins β4 hydrolyzed fragments, up-regulate the expression of calpain 2, and also increase the hydrolysis degree of integrins β4, and at the same time the phosphorylation of Src Y418 site was increased. Conclusion Under high-density culture conditions, E2 can promote the limited hydrolysis of integrin β4, increase the interaction between integrin β4 and Her2, phosphorylate and activate Src, and foster the migration and invasion of SK-Br3 cells by upregulating and activating calpain2.

关键词(KeyWords)： 乳腺肿瘤;雌二醇;人表皮生长因子受体2;钙激活中性蛋白酶2
breast cancer;estradiol(E2);human epidermal growth factor receptor 2(Her2);calcium-activated neutral protease 2(calpain 2)

Abstract:

Keywords:

基金项目(Foundation): 贵州省科学技术基金项目(黔科合J字LKM[2013]02);贵州省科学技术基金项目(黔科合LH字[2016]7351)

作者(Author): 杨琼,王婧雅,王璐,周培富,何可群,严敏,毛大华,陈腾祥,张金娟
YANG Qiong,WANG Jingya,WANG Lu,ZHOU Peifu,HE Kequn,YAN Min,MAO Dahua,CHEN Tengxiang,ZHANG Jinjuan

DOI: 10.19367/j.cnki.2096-8388.2023.02.004

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