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. 2024 May 20;27(5):337-344.
doi: 10.3779/j.issn.1009-3419.2024.101.13.

[TRIM21 Inhibits the Proliferation and Migration of Lung Adenocarcinoma Cells by Interacting with ZSWIM1]

[Article in Chinese]
Luxuan Chen #  1   2 , Qionghua Lian #  1 , Gui Zhang  1 , Jiayao Wu  1 , Guandi Zeng  1 , Xuejuan Gao  1
Affiliations

[TRIM21 Inhibits the Proliferation and Migration of Lung Adenocarcinoma Cells by Interacting with ZSWIM1]

[Article in Chinese]
Luxuan Chen et al. Zhongguo Fei Ai Za Zhi. .

Abstract
in English, Chinese

Background: Lung adenocarcinoma (LUAD) is a highly morbid and fatal cancer. Despite advancements in modern medical treatment, the 5-year survival rate of patients remains suboptimal. Our previous study revealed that zinc finger SWIM-type containing 1 (ZSWIM1), a novel protein, promotes the proliferation, migration, and invasion of LUAD cells. The aim of this study is to investigate the impact of E3 ubiquitin ligase tripartite motif protein 21 (TRIM21) on ZSWIM1-mediated cell proliferation and migration.

Methods: The interaction and co-localization between TRIM21 and ZSWIM1 were verified using co-immunoprecipitation (Co-IP) and immunofluorescence (IF). The effects of TRIM21 and ZSWIM1 on the proliferation and migration of LUAD cells were assessed through MTT and Transwell assays, respectively. Western blot (WB) analysis was conducted to evaluate the impact of TRIM21 and ZSWIM1 on the expression of epithelial-mesenchymal transition (EMT) markers in LUAD cells. The influence of TRIM21 on the ubiquitination of ZSWIM1 was examined using Co-IP combined with WB.

Results: TRIM21 was found to interact and co-localize with ZSWIM1. Overexpression of TRIM21 inhibited the proliferation and migration of LUAD cells. Overexpression of TRIM21 reduced the promoting effect of ZSWIM1 on the proliferation, migration, and invasion of lung adenocarcinoma cells, and reversed the impact of ZSWIM1 on the expression of E-cadherin and Vimentin. Conversely, knockdown of TRIM21 further enhanced the promoting effect of ZSWIM1 on the proliferation and migration of LUAD cells. Mechanistically, we observed that overexpression of TRIM21 significantly enhanced the ubiquitination level of ZSWIM1, leading to a decrease in ZSWIM1 protein expression.

Conclusions: TRIM21 binds to and promotes the ubiquitination of ZSWIM1, resulting in reduced protein expression of ZSWIM1, which leads to the inhibition of ZSWIM1-mediated promotion of proliferation, migration, and invasion in LUAD cells.

【中文题目：TRIM21通过与ZSWIM1相互作用 调节肺腺癌细胞的增殖和迁移】 【中文摘要：背景与目的 肺腺癌（lung adenocarcinoma, LUAD）是一种致病率和死亡率都极高的癌症，尽管现代医学的治疗手段在不断进步，但患者的5年生存率仍不高，因此我们想通过探究LUAD发生发展的分子机制进而鉴定新的治疗靶标。我们前期的研究报道了ZSWIM1（zinc finger SWIM-type containing 1）是一个促进LUAD细胞增殖、迁移、侵袭的新蛋白。本研究将着重探究E3泛素连接酶TRIM21（tripartite-motif protein 21）对ZSWIM1在LUAD细胞中促增殖、迁移功能的影响。方法 利用蛋白免疫共沉淀技术（co-immunoprecipitation, Co-IP）和免疫荧光技术（immunofluorescence, IF）验证TRIM21和ZSWIM1之间的相互作用和共定位；利用MTT和Transwell实验检测TRIM21及TRIM21、ZSWIM1协同对LUAD增殖、迁移的影响；利用蛋白印迹实验（Western blot, WB）检测TRIM21和ZSWIM1对LUAD细胞上皮间充质转化（epithelial-mesenchymal transition, EMT）标志物表达的影响；利用Co-IP联合WB检测TRIM21对ZSWIM1泛素化的影响。结果 TRIM21与ZSWIM1存在相互作用和共定位。TRIM21的上调可以抑制LUAD细胞的增殖和迁移。过表达TRIM21能够降低ZSWIM1对LUAD细胞增殖、迁移和侵袭的促进作用，并逆转ZSWIM1对E-cadherin和Vimentin表达的影响。敲低TRIM21则可以增强ZSWIM1对LUAD细胞增殖和迁移的促进作用。机制方面，我们发现过表达TRIM21可明显增强ZSWIM1的泛素化水平，下调ZSWIM1的蛋白表达量。结论 TRIM21通过结合并促进ZSWIM1的泛素化，进而降低ZSWIM1的蛋白表达，这抑制了ZSWIM1对LUAD细胞增殖、迁移、侵袭表型的促进作用。 】 【中文关键词：肺肿瘤；TRIM21；ZSWIM1；增殖；迁移】.

Keywords: Lung neoplasms; Migration; Proliferation; TRIM21; ZSWIM1.

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Conflict of interest statement

Competing interests: The authors declare that they have no competing interests.

Figures

Fig 1
图 1. 在肺腺癌细胞中TRIM21与ZSWIM1存在相互作用。A：在稳定过表达FLAG-ZSWIM1的细胞中瞬转HA-TRIM21，利用FLAG抗体进行Co-IP实验；B：在H1299细胞中对内源的ZSWIM1和TRIM21进行染色并在共聚焦显微镜下观察，其中ZSWIM1为绿色，TRIM21为红色，细胞核为蓝色，箭头：TRIM21和ZSWIM1共定位的位置。比例尺依次为20、10和5 μm。
Fig 2
图 2. TRIM21抑制肺腺癌细胞中的增殖、迁移功能。A：利用WB实验检测稳定过表达TRIM21的H1299细胞株中TRIM21的表达情况；B：在稳定过表达TRIM21的H1299细胞株中，利用MTT实验检测细胞的增殖能力（n=3, **P<0.01）；C、D：利用Transwell实验检测过表达TRIM21细胞的迁移能力（n=3, **P<0.01）；E：在H1299中瞬转TRIM21的3条siRNA片段，利用WB实验检测TRIM21的表达情况；F：在H1299中敲低TRIM21，利用MTT实验检测细胞的增殖能力（n=3, **P<0.01, ***P<0.001）；G、H：在H1299中敲低TRIM21，利用Transwell实验检测细胞的迁移能力（n=3, **P<0.01）。
Fig 3
图 3. 干扰TRIM21的表达可增强ZSWIM1诱导的H1299细胞增殖、迁移。在稳定过表达ZSWIM1的H1299细胞中敲低TRIM21，A：利用WB实验检测TRIM21的表达情况；B：利用MTT实验检测细胞的增殖能力（n=3, **P<0.01, ***P<0.001）；C、D：利用Transwell实验检测细胞的迁移能力（n=3, ***P<0.001）。
Fig 4
图 4. 过表达TRIM21可减缓ZSWIM1对H1299细胞增殖、迁移、侵袭的促进作用。A、B：在稳定过表达ZSWIM1（OE-FLAG-ZSWIM1）的H1299细胞中瞬转HA-TRIM21，利用Transwell实验检测细胞的迁移、侵袭能力（n=3, **P<0.01, ***P<0.001）；C：在稳定过表达ZSWIM1的H1299细胞中瞬转HA-TRIM21，利用MTT实验检测细胞的增殖能力（n=3, ***P<0.001）；D：同时在H1299细胞中过表达ZSWIM1和TRIM21，利用WB检测EMT标志物E-cadherin、Vimentin。
Fig 5
图 5. 过表达TRIM21可增强ZSWIM1的泛素化。A：在H1299细胞中瞬转FLAG-TRIM21，利用WB检测ZSWIM1蛋白的表达情况；B：在稳定过表达ZSWIM1的H1299细胞中瞬转HA-TRIM21和HA-Ub，并用MG132（20 μmol/L）处理细胞，利用Co-IP技术检测ZSWIM1的泛素化情况。
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