Abstract:

Objective To investigate the molecular mechanism of neddylation UBC12 regulating esophageal cancer cell proliferation. Methods Proteomic analysis was used to screen for downstream targets of UBC12. The expression of RING1 in esophageal cancer patients was analyzed based on the Cancer Genome Atlas（TCGA）database. Esophageal cancer cells with UBC12 knockdown（UBC12 knockdown group）and negative control（NC）cells（NC group），as well as esophageal cancer cells overexpressing（OE）RING1（OE group）and mock（MOCK）-transfected control cells（MOCK group），were established. The effects of RING1 on esophageal cancer cell proliferation were assessed using CCK-8 and colony formation assays. High-throughput sequencing was performed to analyze differentially expressed genes between OE and MOCK groups，followed by bioinformatic analysis. The expression levels of RING1 and UBC12 mRNA in esophageal cancer cells were determined. Protein degradation assays were conducted to analyze the regulatory mechanism of UBC12 on RING1 protein. Cancer tissues and adjacent normal tissues（2 cm from tumor margins） were obtained from 30 esophageal cancer patients at Zhongshan Hospital of Fudan University between January 2017 and December 2018. Immunohistochemical staining was used to determine RING1 protein expression，and all the patients were followed up until March 2025. Kaplan-Meier survival curves were used to analyze patient survival. Results TCGA analysis results showed that RING1 expression was lower in cancer tissues compared to adjacent normal tissues（P<0.001）. Esophageal cancer patients with high RING1 expression exhibited longer overall survival and disease-free survival than those with low RING1 expression. Both cell proliferation viability and the number of cell colonies formed were lower in OE group than those in MOCK group（P<0.05）. Differentially expressed genes between OE and MOCK groups were enriched in biological processes such as axon development and axonogenesis，cellular components including ciliary base and chromatin-remodeling complex，molecular functions such as FAD binding and SMAD binding，pathways including aminoacyl-tRNA biosynthesis，oxidative phosphorylation，RNA transport，protein export and eukaryotic ribosome biogenesis were suppressed in OE group. Compared to NC group，RING1 protein expression was increased in UBC12 knockdown group，while no statistically significance was observed in RING1 mRNA relative expression（P>0.05）. The relative expression of UBC12 mRNA was higher than that of RING1 mRNA in 5 esophageal cancer cell lines（EC1，EC109，K30，K450 and K510）（P<0.05），and UBC12 protein expression was negatively correlated with RING1 protein expression（r=-0.7085，P=0.18）. With prolonged cycloheximide（CHX） treatment，RING1 protein expression in UBC12 knockdown group gradually became higher than in NC group，indicating an extended RING1 protein half-life. RING1 protein expression was lower in cancer tissues than in adjacent normal tissues from esophageal cancer patients（P<0.05）. Esophageal cancer patients with high RING1 expression had a longer overall survival than those with low RING1 expression（Log-rank χ²=4.188，P=0.040 7）. Conclusions UBC12 promotes the proliferation of esophageal cancer cells by regulating RING1，suggesting that it may serve as a novel therapeutic target for esophageal cancer.

Key words: Neddylation UBC12, RING1, Esophageal cancer