Abstract: Objective To explore the mechanism of ARH3 promoting cell proliferation in glioblastoma. Methods The mRNA and protein expression levels of ARH3 in glioblastoma were analyzed by GEPIA2 and CPTAC databases. Two siRNA were designed and transferred either into glioblastoma cell lines U87 and LN229 to silence the expression of ARH3. Western Blot was employed to verify the knockdown efficiency of ARH3. Cell proliferation was detected by clonal formation. Flow cytometry was used to detect the proportion of cells at different periods. EdU assay was used to detect the proportion of cells in S phase. The overexpression vector of Flag-tag ARH3 was constructed and transfected into cells. The interaction proteomics of ARH3 was analyzed by co-immunoprecipitation combined with mass spectrometry and the enrichment analysis of related pathway network was performed. The effect of ARH3 on the stability of cycle-related proteins was verified by cell transfection siRNA combined with Western Blot. The changes of signal pathway network in glioblastoma cells after ARH3 silencing were analyzed by cell transfection siRNA combined mass spectrometry. Results The results of GEPIA2 and CPTAC databases showed that glioblastoma significantly overexpressed ARH3 （P<0.05）; after knocking down endogenous ARH3 in glioblastoma U87 and LN229, the number of colony formation decreased （P<0.05）; the result of flow cytometry showed that the proportion of ARH3 knockdown cells in G1 phase increased （P<0.05）, and the proportion of cells in S phase decreased （P<0.05）; the results of EdU assay showed that the proportion of cells in S phase decreased （P<0.05）; Co-immunoprecipitation combined with mass spectrometry showed the interaction between cell cycle associated proteins and ARH3. Western Blot results showed that the silencing of ARH3 decreased the protein expression level of MCMs and inhibited the expression level of cyclins in S phase. Mass spectrometry showed that ARH3 depression down-regulated the expression of cell cycle-related pathway proteins. Conclusion ARH3 is highly expressed in glioblastoma; ARH3 promotes the transition of glioblastoma in G1/S phase, thereby maintaining the normal progress of cell cycle and promoting the proliferation ability of glioblastoma cells.

Key words: glioblastoma, ARH3, cell cycle, cell Proliferation, MCM2-7

摘要： 目的 探讨ADP-核糖水解酶ARH3在神经胶质母细胞瘤内促进细胞增殖的机制。方法 通过GEPIA2和CPTAC数据库分析ARH3在胶质母细胞瘤中的mRNA及蛋白水平的表达量;设计两条siRNA并分别转入胶质母细胞瘤细胞系U87和LN229细胞内沉默ARH3的表达,利用Western Blot验证ARH3的敲低效率;克隆形成检测细胞增殖情况;流式细胞术检测不同时期的细胞比例;利用EdU实验检测细胞处于S期的比例;构建Flag标签的ARH3过表达载体并转染进入细胞,通过免疫共沉淀技术结合质谱分析ARH3的相互作用蛋白组并进行相关通路网络富集分析;通过细胞转染siRNA结合Western Blot验证ARH3对细胞周期相关蛋白稳定性的影响;采用细胞转染siRNA结合质谱技术分析ARH3沉默后胶质母细胞瘤细胞内部的信号通路网络变化。结果 GEPIA2和CPTAC数据库结果显示胶质母细胞瘤显著高表达ADP-核糖水解酶ARH3（P<0.05）;在胶质母细胞瘤U87和LN229内敲低内源ARH3后,克隆形成数目减少（P<0.05）;流式结果显示内源性ARH3敲降细胞G1期比例增加（P<0.05）,S期细胞比例减少（P<0.05）;EdU实验结果显示S期细胞的比例减少（P<0.05）;免疫共沉淀联合质谱结果鉴定显示细胞周期相关蛋白与ARH3存在相互作用;Western Blot结果显示ARH3的沉默明显降低MCM2-7的蛋白水平,抑制S期周期蛋白表达量;质谱结果显示ARH3的缺失下调了细胞周期相关通路蛋白的表达量。结论 ADP-核糖水解酶ARH3在胶质母细胞瘤内高表达;ARH3通过促进胶质母细胞瘤在G1/S期的转换,从而维持细胞周期的正常进行,促进细胞的增殖能力。

关键词: 胶质母细胞瘤, ARH3, 细胞周期, 细胞增殖, MCM2-7