RAC1通过调节紧密连接蛋白1分布影响卵巢癌细胞的迁移和侵袭  

RAC1 inhibits the migration and invasion of human epithelial ovarian cancer cells by regulating the distribution of tight junction protein 1

作　　者：张璐玢 邹雪琴 张鳅丹 高淑君 刘雪 潘玲丽 赵杨静[1] 梁秀婷 王荟[1,3] 朱彦玲 邵启祥[1,3] ZHANG Lu-bin;ZOU Xue-qin;ZHANG Qiu-dan;GAO Shu-jun;LIU Xue;PAN Ling-li;ZHAO Yang-jing;LIANG Xiu-ting;WANG Hui;ZHU Yan-ling;SHAO Qi-xiang(School of Medicine,Jiangsu University,Zhenjiang Jiangsu 212013;Department of Gynecology and Obstetrics,Xuzhou Hospital Affiliated to Jiangsu University,Xuzhou Jiangsu 221005;Key Laboratory of Medical Science and Laboratory Medicine of Jiangsu Province,Zhenjiang Jiangsu 212013,China)

机构地区：[1]江苏大学医学院,江苏镇江212013 [2]江苏大学附属徐州医院妇产科,江苏徐州221005 [3]江苏省检验医学重点实验室,江苏镇江212013

出　　处：《江苏大学学报（医学版）》2020年第5期408-413,共6页Journal of Jiangsu University：Medicine Edition

基　　金：国家自然科学基金资助项目(31400773,81671541);徐州市重点研发计划(社会发展)项目(KC19147);江苏省大学生创新创业训练计划项目(201910299156Y,201710299063Y,201810299089Y)。

摘　　要：目的:探讨小GTP酶Rac1(Rac family small GTPase 1,RAC1)对卵巢癌细胞迁移和侵袭的影响及机制。方法:构建pLKO.1-puro RAC 1 shRNA载体,包装慢病毒,感染卵巢癌细胞OVCAR3和SKOV3,并采用嘌呤霉素筛选;通过蛋白质印迹、细胞划痕和基质胶侵袭实验分别检测细胞中RAC 1敲减效率,细胞迁移和侵袭能力的改变;采用蛋白质印迹和免疫荧光染色观察细胞中紧密连接蛋白1(tight junction protein 1,TJP1)的表达和分布变化。结果:酶切电泳和测序结果均显示pLKO.1-puro RAC 1 shRNA慢病毒载体构建成功,蛋白质印迹结果证实感染RAC 1 shRNA慢病毒的OVCAR3和SKOV3细胞中RAC1蛋白水平较对照组明显下降(P<0.05)。细胞划痕和基质胶侵袭实验证实,RAC 1敲减后卵巢癌细胞的迁移和侵袭能力较对照组显著下降(P均<0.01);蛋白质印迹结果显示RAC 1敲减后卵巢癌细胞中TJP1蛋白表达水平无明显变化(P>0.05),但免疫荧光染色表明TJP1由胞质内散在分布转变为细胞间连接处聚集分布,细胞间连接更加紧密。结论:RAC 1基因可能通过改变TJP1蛋白分布从而影响卵巢癌细胞的迁移和侵袭。Objective:To investigate the effects and mechanism of Rac family small GTPase 1(RAC1)on the migration and invasion of ovarian cancer cells.Methods:The pLKO.1-puro RAC 1 shRNA lentiviral vector was constructed to package lentivirus and infect ovarian cancer cell lines OVCAR3 and SKOV3 which were selected by puromycin.Western blotting was performed to determine the RAC1 knockdown efficiency.Wound healing assay and matrigel invasion assay were applied to detect the migration and invasion.The protein expression and distribution of tight junction protein 1(TJP1)were visualized by Western blotting and immunefluorescence staining.Results:Restriction enzyme digestion and sequencing confirmed the successful construction of RAC 1 shRNA lentiviral vector.Western blotting showed the stable RAC 1 knockdown in OVCAR3 and SKOV3(P<0.05).After knocking down RAC 1,the cell migration and invasion were significantly decreased(all P<0.01).Western blotting confirmed that RAC 1 knockdown in OVCAR3 and SKOV3 did not affect TJP1 protein expression,but relocated TJP1 distribution from the cytoplasm to cell-cell junction and strengthened the connection between cells.Conclusion:RAC 1 knockdown might inhibit the migration and invasion of ovarian cancer cells by regulating the intercellular distribution of TJP1.

关 键 词：小GTP酶Rac1 紧密连接蛋白1 迁移 侵袭 卵巢癌 

分 类 号：R737.31[医药卫生—肿瘤]