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

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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 RAC1 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RAC1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 RAC1 shRNA慢病毒载体构建成功，蛋白质印迹结果证实感染RAC1 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，RAC1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RAC1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： RAC1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。

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	张璐玢1
	邹雪琴1
	张鳅丹1
	高淑君1
	刘雪1
	潘玲丽1
	赵杨静1
	梁秀婷2
	王荟1
	3
	朱彦玲2
	邵启祥1
	3

关键词 ：小GTP酶Rac1, 紧密连接蛋白1, 迁移, 侵袭, 卵巢癌

Abstract：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 RAC1 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 RAC1 shRNA lentiviral vector. Western blotting showed the stable RAC1 knockdown in OVCAR3 and SKOV3 (P<0.05). After knocking down RAC1, the cell migration and invasion were significantly decreased (all P<0.01). Western blotting confirmed that RAC1 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: RAC1 knockdown might inhibit the migration and invasion of ovarian cancer cells by regulating the intercellular distribution of TJP1.

收稿日期: 2020-04-16

基金资助: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）

通讯作者: 邵启祥（通讯作者），教授，博士生导师，E-mail：shao_qx@ ujs.edu.cn

作者简介: 张璐玢(1994—)，女，硕士研究生

引用本文:

张璐玢1，邹雪琴1，张鳅丹1，高淑君1，刘雪1，潘玲丽1，赵杨静1，梁秀婷2，王荟1,3，朱彦玲2，邵启祥1,3. 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[J]. 江苏大学学报:医学版, 2020, 30(05): 408-413.

链接本文:

http://zzs.ujs.edu.cn/xbyxb/CN/ 或 http://zzs.ujs.edu.cn/xbyxb/CN/Y2020/V30/I05/408

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