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

doi:10.12122/j.issn.1673-4254.2026.09.12

Abstract

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

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.

Figures/Tables 6

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.

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).

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.

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.

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.

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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