Background: As an important deubiquitinating enzyme, Ubiquitin Carboxyl-terminal Hydrolase L3 (UCHL3) has been considered to play an important role in tumor progression and DNA damage repair. However, its specific function and regulatory mechanism in colorectal cancer remain to be fully elucidated. This study aims to explore the functional role of deubiquitinating enzyme UCHL3 in colorectal cancer, focusing on analyzing its specific mechanism in DNA damage repair and cell cycle regulation by regulating Cell Division Cycle Associated 5 (CDCA5) protein stability, thereby revealing the role of UCHL3 in colorectal cancer and its potential impact on cancer progression.

Methods: The experiment consists of normal colon epithelial cells (FHC) and colorectal cancer cell line (SW480) as research subjects to construct UCHL3 knockdown and overexpression models, respectively. The expression levels of UCHL3 and CDCA5 were evaluated using quantitative real-time PCR (qRT-PCR) and Western blot analyses, respectively. Cell viability was assessed using the CCK-8 assay, apoptosis rate and cell cycle distribution were analyzed using flow cytometry, DNA damage level was assessed via comet assay, and Co-IP was used to verify the relationship between UCHL3 and CDCA5, protein interaction of CDCA5, and its ubiquitination level of CDCA5.

Results: UCHL3 and CDCA5 were significantly upregulated in CRC cells compared with normal controls (p < 0.05). Silencing UCHL3 markedly reduced cell viability (p < 0.05), increased apoptosis (p < 0.05), enhanced DNA damage (p < 0.05), and induced G2/M cell cycle arrest (p < 0.05). Conversely, UCHL3 overexpression significantly promoted cell viability (p < 0.05), decreased apoptosis (p < 0.05), and attenuated DNA damage (p < 0.05). Mechanistically, UCHL3 stabilized CDCA5 protein by suppressing its ubiquitination (p < 0.05), thereby facilitating DNA damage repair and regulating cell cycle progression.

Conclusion: UCHL3 promotes DNA damage repair and inhibits apoptosis by stabilizing CDCA5, thereby driving the progression of colorectal cancer. UCHL3 may become a potential target for treating colorectal cancer.