Nan fang yi ke da xue xue bao = Journal of Southern Medical University · 2026
[The mitochondrial protein encoded by circUSP25 promotes colon cancer cell proliferation and migration].
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Li Xiaomin, Xu Jie, Gao Caiyue, Zhu Jijun, Wang Yu, Li Dongdong, Wang Xiaoyan
Аннотация
OBJECTIVES: 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.
CONCLUSIONS: The circUSP25-encoded protein localizes to the mitochondria, and its overexpression promotes colon cancer cell proliferation and migration possibly by interacting with mitochondrial proteins.
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.
CONCLUSIONS: The circUSP25-encoded protein localizes to the mitochondria, and its overexpression promotes colon cancer cell proliferation and migration possibly by interacting with mitochondrial proteins.