背景:MutT同源物1(MTH1)对氧化的dNTP池进行消毒以促进癌细胞的存活,并且其表达在癌症中经常上调。聚泛素化稳定MTH1以促进黑色素瘤细胞的增殖,表明泛素系统控制MTH1的稳定性和功能。然而,泛素化是否调节胃癌中的MTH1尚未得到很好的定义。本研究旨在探讨MTH1与去泛素酶之间的相互作用,USP9X,在调节增殖方面,生存,迁移,和胃癌细胞的侵袭。
方法:在HGC-27胃癌细胞中通过共免疫沉淀(co-IP)评估了USP9X与MTH1之间的相互作用。siRNA用于干扰胃癌细胞系HGC-27和MKN-45中的USP9X表达。进行MTT测定以检查增殖,碘化丙啶(PI)和7-AAD染色测定,以评估细胞周期,进行膜联蛋白V/PI染色测定以检查细胞凋亡,和transwell分析用于确定对照的迁移和入侵,缺乏USP9X,和USP9X缺陷加上MTH1过表达的HGC-27和MKN-45胃癌细胞。
结果:Co-IP数据显示USP9X与MTH1相互作用并去泛素化。USP9X的过表达通过下调其泛素化提高MTH1蛋白水平,而USP9X的敲低对MTH1有相反的影响。HGC-27和MKN-45细胞中的USP9X缺乏导致增殖减少,细胞周期停滞,额外的凋亡,以及有缺陷的迁移和入侵,可以通过过量的MTH1来拯救。
结论:USP9X与MTH1相互作用并稳定MTH1以促进其增殖,生存,胃癌细胞的迁移和侵袭。
BACKGROUND: MutT homolog 1 (MTH1) sanitizes oxidized dNTP pools to promote the survival of cancer cells and its expression is frequently upregulated in cancers. Polyubiquitination stabilizes MTH1 to facilitate the proliferation of melanoma cells, suggesting the ubiquitin system controls the stability and function of MTH1. However, whether ubiquitination regulates MTH1 in gastric cancers has not been well defined. This study aims to investigate the interaction between MTH1 and a deubiquitinase, USP9X, in regulating the proliferation, survival, migration, and invasion of gastric cancer cells.
METHODS: The interaction between USP9X and MTH1 was evaluated by co-immunoprecipitation (co-IP) in HGC-27 gastric cancer cells. siRNAs were used to interfere with USP9X expression in gastric cancer cell lines HGC-27 and MKN-45. MTT assays were carried out to examine the proliferation, propidium iodide (PI) and 7-AAD staining assays were performed to assess the cell cycle, Annexin V/PI staining assays were conducted to examine the apoptosis, and transwell assays were used to determine the migration and invasion of control, USP9X-deficient, and USP9X-deficient plus MTH1-overexpressing HGC-27 and MKN-45 gastric cancer cells.
RESULTS: Co-IP data show that USP9X interacts with and deubiquitinates MTH1. Overexpression of USP9X elevates MTH1 protein level by downregulating its ubiquitination, while knockdown of USP9X has the opposite effect on MTH1. USP9X deficiency in HGC-27 and MKN-45 cells causes decreased proliferation, cell cycle arrest, extra apoptosis, and defective migration and invasion, which could be rescued by excessive MTH1.
CONCLUSIONS: USP9X interacts with and stabilizes MTH1 to promote the proliferation, survival, migration and invasion of gastric cancer cells.