Effect of USP1 on Proliferation and Cell Cycle of Bladder Cancer Cells by Regulating E2F1
PENG Peng, ZHANG Anqi, ZHOU Manli, LIU Feiliu, ZHANG Huihui, ZHANG Xia*
This study aimed to investigate the effect of USP1 (ubiquitin-specific Protease 1) on the biological behavior of bladder cancer cells such as proliferation and cell cycle, and further explore its mechanism. The over-expression of USP1 in bladder cancer T24 cell line was established by molecular cloning technique, the USP1 knockout cell line of bladder cancer UMUC3 cell line was established by CRISPR-Cas9 technique, the cell proliferation and clonogenic ability were detected by CCK8 and clonogenic test, cell migration was detected by scratch test, cell cycle was detected by flow cytometry with PI staining. The difference of gene expression was detected by transcriptome sequencing, and the effect of USP1 on signal transduction were measured by dual luciferase reporter gene and then verified by immunoblotting. The results showed that overexpression of USP1 could promote the proliferation of bladder cancer cells, and knockout of USP1 significantly inhibited the proliferation, colony formation and migration of bladder cancer cells, and promoted s-phase cell arrest. The results from RNA sequence showed that there were 4 522 differentially expressed genes between USP1 knockout cells and wide type cells. Among them, 2 078 genes expression were up-regulated and 2 444 genes expression were down-regulated, which mainly involved in cell cycle regulation, signal transduction, transcription and translation, protein folding and degradation, autophagy and apoptosis, etc. The results of Hallmark database analysis showed that differentially expressed genes were closely related to E2F signaling pathway. Moreover, dual luciferase reporter genes also showed that the E2F1 signaling pathway was up-regulated in a dose-dependent manner after USP1 overexpression. Immunoblotting results also showed that E2F1 protein level was down-regulated after USP1 knockout. This study revealed that USP1 promotes bladder cancer progression by regulating the proliferation and cycle of bladder cancer cells through E2F1.
CN
EN