circUSP25编码的线粒体蛋白促进结肠癌细胞增殖及迁移

doi:10.12122/j.issn.1673-4254.2026.09.12

南方医科大学学报 ›› 2026, Vol. 46 ›› Issue (9): 2123-2129.doi: 10.12122/j.issn.1673-4254.2026.09.12

• • 上一篇

circUSP25编码的线粒体蛋白促进结肠癌细胞增殖及迁移

李晓敏¹^,²(), 徐洁¹^,², 高彩月²^,³, 朱季军¹^,², 王玉²^,³, 李冬冬²^,³, 王晓燕¹^,²()

^1.江苏省人民医院宿迁医院，消化内科，江苏宿迁 223800
^2.江苏省人民医院宿迁医院，宿迁市结直肠癌及炎症性肠病基础与临床转化重点实验室，江苏宿迁 223800
^3.江苏省人民医院宿迁医院，临床医学研究中心，江苏宿迁 223800

收稿日期:2026-02-28 出版日期:2026-09-20 发布日期:2026-09-30
通讯作者: 王晓燕 E-mail:lxm1980326@sina.com;17805296777@163.com
作者简介:李晓敏，副研究员，硕士生导师，E-mail: lxm1980326@sina.com
基金资助:
国家自然科学基金(82573244);国家自然科学基金(81902479);江苏省卫健委医学科研项目(M2024038);广东省基础与应用基础研究基金(2021A1515010665);宿迁市科技计划(KY202304);宿迁市科技计划(M202503);宿迁英才雄英计划(SQXY202436)

The mitochondrial protein encoded by circUSP25 promotes colon cancer cell proliferation and migration

Xiaomin LI¹^,²(), Jie XU¹^,², Caiyue GAO²^,³, Jijun ZHU¹^,², Yu WANG²^,³, Dongdong LI²^,³, Xiaoyan WANG¹^,²()

^1.Department of Gastroenterology, Jiangsu Province (Suqian) Hospital, Suqian 223800, China
^2.Suqian Key Laboratory of Basic and Clinical Translation for Colorectal Cancer and Inflammatory Bowel Disease, Jiangsu Province (Suqian) Hospital, Suqian 223800, China
^3.Clinical Medical Research Center, Jiangsu Province (Suqian) Hospital, Suqian 223800, China

Received:2026-02-28 Online:2026-09-20 Published:2026-09-30
Contact: Xiaoyan WANG E-mail:lxm1980326@sina.com;17805296777@163.com
Supported by:
National Natural Science Foundation of China(82573244)

摘要/Abstract

摘要：

目的探索circUSP25编码蛋白的生物学功能及其潜在机制。方法通过circRNADb和circBank数据库预测circUSP25的编码潜能；采用Sanger测序验证circUSP25的剪接位点；构建circUSP25-Flag野生型及起始密码子ATG突变载体，转染结肠癌细胞SW620和HCT116，应用Western blotting、免疫沉淀、质谱及免疫荧光鉴定circUSP25编码的蛋白及其亚细胞定位；运用TargetP-2.0数据库预测蛋白的信号肽；采用CCK-8和Transwell小室迁移实验评估该蛋白对结肠癌细胞增殖及迁移的影响；通过免疫共沉淀及质谱筛选其相互作用蛋白，免疫荧光验证其与候选蛋白PHB2的共定位关系；应用CCK-8和Transwell小室迁移实验检测敲低PHB2对circUSP25编码蛋白促进增殖及迁移作用的回复。结果 circUSP25由USP25基因第2、3外显子背向剪接形成，含有编码121个氨基酸的开放阅读框。Western blotting及质谱鉴定证实circUSP25可编码蛋白，该蛋白定位于结肠癌细胞的线粒体。TargetP-2.0预测显示其氨基端1-32aa内含有线粒体转运肽。过表达circUSP25编码蛋白可显著促进结肠癌细胞增殖及迁移（P<0.05）。免疫共沉淀联合质谱鉴定发现该蛋白可与线粒体蛋白PHB2等结合，免疫荧光证实二者在线粒体存在共定位，功能挽救实验表明敲低PHB2可以回复circUSP25编码蛋白对结肠癌细胞增殖及迁移的作用。结论 circUSP25编码的蛋白定位于线粒体，可能通过与线粒体蛋白相互作用促进结肠癌细胞增殖与迁移。

关键词: 结肠癌, 环状RNA, circUSP25, hsa_circ_0001178, 线粒体蛋白

Abstract:

Objective To investigate the regulatory role of the protein encoded by circUSP25 in proliferation and migration of colon cancer cells. Methods The coding potential of circUSP25 was predicted using circRNADb and circBank databases, and the splice junction was validated by Sanger sequencing. circUSP25-Flag wild-type vector and mutant vector with start codon ATG mutation were constructed and transfected into SW620 and HCT116 colon cancer cells, and the expression of the encoded protein and its subcellular localization were analyzed using Western blotting, immunoprecipitation, mass spectrometry, and immunofluorescence staining. The signal peptide was predicted using TargetP-2.0. The effects of this protein on proliferation and migration of colon cancer cells were evaluated using CCK8 and Transwell migration assays. The interacting proteins were screened by co-immunoprecipitation coupled with mass spectrometry, and its co-localization with the candidate protein PHB was verified by immunofluorescence staining. The rescue effect of PHB2 knockdown on the activity of circUSP25-encoded protein for promoting colon cancer cell proliferation and migration were observed. Results circUSP25 was formed by back-splicing of exons 2 and 3 of the parental gene USP25 and contained an open reading frame encoding 121 amino acids. circUSP25 encodes a protein localizing to the mitochondria of colon cancer cells, and its N-terminal 1-32 amino acids contain a mitochondrial transit peptide. Overexpression of the circUSP25-encoded protein significantly promoted colon cancer cell proliferation and migration. Co-immunoprecipitation coupled with mass spectrometry revealed that this protein binds to the mitochondrial proteins such as PHB2, and they co-localize in the mitochondria. Functional rescue experiments showed that PHB2 knockdown reversed the pro-proliferative and pro-migratory effects of this circUSP25-encoded protein. Conclusion The circUSP25-encoded protein localizes to the mitochondria, and its overexpression promotes colon cancer cell proliferation and migration possibly by interacting with mitochondrial proteins.

Key words: colon cancer, circular RNA, circUSP25, hsa_circ_0001178, mitochondrial proteins

李晓敏, 徐洁, 高彩月, 朱季军, 王玉, 李冬冬, 王晓燕. circUSP25编码的线粒体蛋白促进结肠癌细胞增殖及迁移[J]. 南方医科大学学报, 2026, 46(9): 2123-2129.

Xiaomin LI, Jie XU, Caiyue GAO, Jijun ZHU, Yu WANG, Dongdong LI, Xiaoyan WANG. The mitochondrial protein encoded by circUSP25 promotes colon cancer cell proliferation and migration[J]. Journal of Southern Medical University, 2026, 46(9): 2123-2129.

图/表 6

图1 环状RNAcircUSP25开放阅读框的预测

Fig.1 Prediction of the open reading frame (ORF) of circUSP25. A: Schematic diagram of the formation of circUSP25, which is generated from back-splicing of exons 2 and 3 of the parental gene USP25. Sanger sequencing confirms that the splice junction sequence of circUSP25 is completely consistent with the head-to-tail splicing of exons 2 and 3 of USP25. B: The ORF of circUSP25 (the green line). C: Schematic diagrams of the wild-type circUSP25-Flag vector containing the translation start codon ATG and the mutant circUSP25-Flag-Mut vector with ATG mutation. D: Sanger sequencing of circUSP25-Flag and circUSP25-Flag-Mut vectors confirms the correct sequences of the translation start codon ATG and its mutation.

图2 circUSP25编码蛋白的鉴定

Fig.2 Identification of the protein encoded by circUSP25. A: Western blotting showing that the circUSP25-Flag vector translates the protein, while the ATG-mutated vector fails to translate. B: Immunoprecipitation coupled with mass spectrometry identifies peptides corresponding to the protein encoded by circUSP25-Flag. C: Immunofluorescence staining reveals that the protein encoded by circUSP25-Flag localizes to the cytoplasm of colon cancer cells (Scale bar=25 μm).

图3 circUSP25编码的蛋白定位于结肠癌细胞的线粒体

Fig.3 The protein encoded by circUSP25 localizes to the mitochondria of colon cancer cells. A: TargetP-2.0 database prediction shows that the N-terminal 1-32 amino acids of circUSP25-encoded protein contain a mitochondrial transit peptide. B: Immunofluorescence staining demonstrates that circUSP25-encoded protein localizes to the mitochondria of colon cancer cells.

图4 circUSP25编码的蛋白促进结肠癌细胞增殖和迁移

Fig.4 The protein encoded by circUSP25 promotes proliferation and migration of colon cancer cells. A: CCK8 assays showing that circUSP25-Flag transfection promotes proliferation of colon cancer cells. B: Transwell assays showing that transfection with circUSP25-Flag enhances migration of colon cancer cells (Scale bar=100 μm). *P<0.05, ***P<0.001, ****P<0.0001 vs NC group.

图5 circUSP25编码的蛋白与线粒体蛋白结合

Fig.5 The protein encoded by circUSP25 binds to mitochondrial proteins. A: Co-immunoprecipitation, electrophoretic separation, and silver nitrate staining reveal a differential band near 35 kD, identified subsequently by mass spectrometry as proteins interacting with the circUSP25-encoded protein. B: Mass spectrometry identifies 138 proteins, among which 15 match the expected molecular weight range. Ranked by confidence and the number of unique peptides, 3 of the top 4 proteins are mitochondrial proteins. C: Top 15 proteins matching the molecular weight range ranked by confidence and the number of unique peptides, with mitochondrial proteins highlighted in red. D: Immunofluorescence staining showing co-localization of the circUSP25-encoded protein with PHB2 in the mitochondria.

图6 敲低PHB2可回复circUSP25编码蛋白对结肠癌细胞增殖和迁移的影响

Fig.6 Knockdown of PHB2 reverses the effects of the circUSP25-encoded protein on colon cancer cell proliferation and migration. A: CCK8 assays showing that PHB2 knockdown reverses the proliferation-promoting effect of the circUSP25-encoded protein. B: Transwell assays showing that PHB2 knockdown reverses the migration-promoting effect of the circUSP25-encoded protein (Scale bars=100 μm). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs circUSP25-Flag.

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circUSP25编码的线粒体蛋白促进结肠癌细胞增殖及迁移

The mitochondrial protein encoded by circUSP25 promotes colon cancer cell proliferation and migration

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