Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes, Institute of Life and Health Engineering, Jinan University, Guangzhou 510632, Guangdong, China.
Center of Kidney Disease, Huadu District People's Hospital, Southern Medical University, Guangzhou 510800, Guangdong, China.
Biomed Environ Sci. 2018 Mar;31(3):186-196. doi: 10.3967/bes2018.024.
Osteosarcoma is the most common type of malignant bone tumor in children and adolescents. The role of E3 ligases in tumorigenesis is currently a focus in tumor research. In the present study, we investigated the role of the E3 ligase tripartite motif 21 (TRIM21) in osteosarcoma cell proliferation.
3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assays were used to assess osteosarcoma cell viability. U2-OS cells stably carrying a recombinant lentivirus expressing tetracycline-regulated TRIM21 were screened. Co-immunoprecipitation was coupled with LCMS/MS analysis to identify novel interacting partners of TRIM21. Co-immunoprecipitation and bimolecular fluorescence complementation (BIFC) were performed to validate the interactions between TRIM21 and its novel partner YWHAZ. A TRIM21-ΔRING construct was generated to test the effects of TRIM21 ligase activity on YWHAZ.
TRIM21 positively regulated osteosarcoma cell proliferation. Overexpression of TRIM21 enhanced osteosarcoma cell tolerance toward various stresses. YWHAZ protein was identified as a novel interacting partner of TRIM21 and its expression levels were negatively regulated by TRIM21. The RING domain of TRIM21 was required for TRIM21 negative regulation of YWHAZ expression. However, overexpression of YWHAZ did not affect positive regulation of osteosarcoma cell proliferation by TRIM21.
Our results further clarify the molecular mechanisms underlying the pathogenesis of osteosarcoma.
骨肉瘤是儿童和青少年中最常见的恶性骨肿瘤类型。E3 连接酶在肿瘤发生中的作用目前是肿瘤研究的一个焦点。在本研究中，我们研究了 E3 连接酶三基序 21（TRIM21）在骨肉瘤细胞增殖中的作用。
使用 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）测定法评估骨肉瘤细胞活力。筛选稳定携带表达四环素调控的 TRIM21 的重组慢病毒的 U2-OS 细胞。将共免疫沉淀与 LCMS/MS 分析相结合，以鉴定 TRIM21 的新相互作用伙伴。进行共免疫沉淀和双分子荧光互补（BIFC）实验以验证 TRIM21 与其新型伙伴 YWHAZ 之间的相互作用。生成 TRIM21-ΔRING 构建体以测试 TRIM21 连接酶活性对 YWHAZ 的影响。
TRIM21 正向调节骨肉瘤细胞增殖。TRIM21 的过表达增强了骨肉瘤细胞对各种应激的耐受能力。YWHAZ 蛋白被鉴定为 TRIM21 的新相互作用伙伴，其表达水平受 TRIM21 负调控。TRIM21 的 RING 结构域是 TRIM21 负调控 YWHAZ 表达所必需的。然而，YWHAZ 的过表达不会影响 TRIM21 对骨肉瘤细胞增殖的正向调节作用。
我们的结果进一步阐明了骨肉瘤发病机制的分子机制。
