【摘要】 该文探讨了泛素特异蛋白酶1(USP1)对膀胱癌细胞增殖和周期等生物学行为的作用,并进一步探索其作用机制。通过分子克隆技术构建膀胱癌T24细胞USP1过表达细胞株; CRISPRCas9技术构建膀胱癌UMUC3细胞USP1敲除细胞株; CCK8和克隆形成实验检测细胞增殖和克隆形成能力;划痕实验检测细胞迁移; PI染色流式细胞术检测细胞周期;转录组测序检测USP1敲除后基因表达差异及其相关的功能与信号通路;双荧光素酶报告基因检测USP1对信号通路的影响,并通过免疫印迹技术进行验证。结果显示, USP1过表达可以促进膀胱癌细胞增殖,敲除后显著抑制了膀胱癌细胞的增殖和克隆形成及迁移能力,促进S期细胞阻滞。转录组测序结果显示, USP1敲除后差异表达基因共4 522个,其中上调基因2 078个,下调基因2 444个。KEGG分析结果显示,这些基因涉及多个方面,包括细胞周期调控、细胞信号转导、转录翻译、蛋白折叠降解、自噬凋亡等。Hallmark数据库分析结果显示,差异表达基因与E2F信号通路密切相关。双荧光素酶报告基因显示, USP1过表达后, E2F1信号通路明显上调并且呈剂量依赖式,免疫印迹结果也显示USP1敲除后E2F1蛋白水平下调明显。该研究得出, USP1通过E2F1调控膀胱癌细胞增殖和周期,从而促进膀胱癌进展。更多还原

【Abstract】 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 USP1knockout 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 E2F1signaling 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 USP1promotes bladder cancer progression by regulating the proliferation and cycle of bladder cancer cells through E2F1.更多还原