摘要
目的观察泛素特异性蛋白酶USP4对转化生长因子-βⅠ型受体（TGFβRI）的去泛素化调节及其对乳腺癌细胞生物学活性的调控。方法通过慢病毒感染构建过表达USP4的稳定乳腺癌细胞株BT-549。在乳腺癌细胞中表达外源性泛素，通过泛素化实验验证USP4对TGFβRI的泛素化修饰。采用克隆形成实验和划痕实验检测USP4对乳腺癌细胞克隆形成能力、迁移能力的影响。结果过表达USP4的稳定乳腺癌细胞株中USP4mRNA的表达量（△Ct=3．43±0．05）是空白对照细胞（△Ct=7．26±0．03）的14．22倍，差异有统计学意义（t=69．350，P=0，000）；USP4蛋白的表达量是空白对照细胞的4．99倍，差异有统计学意义（t=28．440，P=0．000）。过表达USP4后TGFβ3RI的体外泛素化水平明显降低。在克隆形成实验中，稳定过表达USP4、病毒对照及空白对照的BT-549细胞克隆形成率分别为（25．83±1．01）％、（11．83±0．60）％、（7．17±0．60）％，分别t=11．880、15．840，差异有统计学意义（P=0．000）。在划痕试验中，放大200倍视野下观察稳定过表达USP4、病毒对照及空白对照的BT-549细胞，每个视野可见发生迁移的细胞个数为（56．67±1．76）、（27．00±1．16）、（27．33±1．45）个，t=14．070、12．840，差异有统计学意义（P=0．000）。结论USP4可通过其去泛素化酶的活性解除TGFβRI耦联的泛素链稳定TGFβRI的表达，从而增强乳腺癌细胞的生物学活性。
Objective To study the deubiquitination regulation of ubiquitin - specific protease USP4 on transforming growth factor - β type Ⅰ receptor （TGFI3RI） and its regulation on biological activity of breast cancer cells. Methods The stable breast cancer cell line BT - 549 with USP4 over - expression was constructed by recombinant lentivirus infection. Ubiqutin - WT - hemagglutinin （HA） - pcDNA was transfected into breast cancer cells through lipofectamine 3000 and the ubiquitination of TGFβRI by USP4 was examined by specific ubiquitination experiments, and overexpression of USP4 was seen to reduce ubiquitin attached by TGFβRI. The colony - forming and migration ability of breast cancer cells were detected by colony formation assay and wound healing assay. Results The USP4 mRNA and protein expression in the cells were detected by fluorescence quantitative polymerase chain reaction and Western blotting, and the relative level of USP4 mRNA in the stable breast cancer cell lines （ ACt = 5.45 ± 0. 05 ） were 14. 22 times respectively compared with the control cells （ ACt = 7.26 ± 0. 03 ）, t = 69. 350, P = 0. 000. And the USP4 protein in the stable breast cancer cell lines were 6. 99 times respectively compared with the control cells, t = 28. 440, P = O. 000. The ubiquitination level of TGFβRI was significantly decreased with USP4 over - expression. In colony formation assay and wound healing assay, the colony formation rates of stably overexpressing USP4 BT- 549 ceils, virus control and blank control were respectively （25.83 ± 1.01 ） %, （ 11.83 ± 0. 60） %, （7.17 ± 0. 60） %, P = 0. 000 and the number of cells that migrated were respectively 56. 67 ± 1.76, 27.00 ± 1.16, and 27.53 ± 1.45, P = 0. 000. Conclusion Our results indicate that USP4 can release the TGFβRI - coupled ubiquitin to stabilize its expression and thus enhance the biological activity of breast cancer cells.
作者
曾响
李月云
刘相萍
迟静薇
刘家秀
周泉
王海波
Zeng Xiang;Li Yueyun;Liu Xiangping;Chi Jingwei;Liu Jiaxiu;Zhou Quan;Wang Haibo(Center of Diagnosis and Treatment of Breast Disease, the Affiliated Hospital of Qingdao University, Qingdao 266003, China （ Zeng X, Li YY, Wang HB;Clinical Medical Research Center, the Affiliated Hospital of Qingdao University, Qingdao 266003, China （ Liu XP, Chi JW, Liu JX, Zhou Q)
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2018年第7期1247-1249,共3页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金（81572616、81772845）
山东省自然科学基金（ZR2017MH016）
