MMS21通过维持MCM2-7复合物稳定性促进肝细胞癌增殖活性  

MMS21 Maintains the Stability of Complex MCM2-7 to Promote Hepatocellular Carcinoma Proliferation

作　　者：李文佳 朱元昕 胡开顺 LI Wen-jia;ZHU Yuan-xin;HU Kai-shun(Medical Research Center,Sun Yat-sen Memorial Hospital//Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation,Guangzhou 510080,China)

机构地区：[1]中山大学孙逸仙纪念医院基础与转化医学研究中心//广东省恶性肿瘤表观遗传与基因调控重点实验室,广东广州510080

出　　处：《中山大学学报（医学科学版）》2022年第2期203-211,共9页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：国家自然科学基金(82172636);广东省恶性肿瘤表观遗传与基因调控重点实验室项目(2020B1212060018)。

摘　　要：【目的】探讨SUMO E3泛素连接酶MMS21在肝细胞癌的表达及发挥作用机制。【方法】利用GEPIA2和CPATC数据库分析MMS21在肝细胞癌中的mRNA和蛋白表达变化及与患者预后的关系;设计两条siRNA分别敲低肝癌细胞内源性MMS21的表达,并采用流式细胞技术检测细胞周期分布比例;进行BrdU染色和DNA fiber实验检测细胞增殖能力与DNA合成速度;构建带有MYC标签的MMS21蛋白过表达载体,并在细胞系SK-hep1中建立其稳定表达的细胞株;利用Co-IP联合质谱分析的方法鉴定MMS21的互作蛋白组并进行信号通路富集分析;采用West⁃ern Blot,检测MMS21对细胞周期相关蛋白的影响。【结果】在肝细胞癌中,与癌旁组织(160例)相比,MMS21在癌组织(369例)中显著高表达(P<0.05),其高表达与患者不良预后相关(P<0.01)。在肝癌细胞系中,使用两条siRNA分别敲低内源MMS21的表达后,SK-hep1细胞中G1期的细胞比例显著上升(P<0.001;P<0.01),S期比例显著下降(P<0.01;P<0.05),G2/M期比例下降(P<0.05;P<0.05);HepG2细胞中G1期的细胞比例显著上升(P<0.001;P<0.001),S期比例显著下降(P<0.01;P<0.01),G2/M期比例下降(P<0.05;P<0.05);SK-hep1细胞的DNA合成速度明显减慢(P<0.0001;P<0.0001);SK-hep1和HepG2细胞增殖能力显著下降(P<0.01;P<0.001;P<0.001;P<0.001)。质谱鉴定MMS21的互作蛋白组,共鉴定到641个高可信度蛋白;通路富集分析表明,这些结合蛋白显著富集于细胞周期相关的生理过程;进一步的Western Blot结果表明:MMS21的缺失能抑制S期和G2/M期的周期蛋白表达量,降低微小染色体维持蛋白家族(MCMs)的蛋白水平。【结论】SUMO E3连接酶MMS21在肝细胞癌中高表达,并与患者不良预后相关;MMS21与肝癌细胞的增殖密切相关,能够维持细胞周期蛋白和复制复合物MCM蛋白稳定性,促进肝癌细胞的增殖能力。【Objective】To investigate the expression level of MMS21 and its functions in hepatocellular carcinoma.【Methods】The GEPIA2 and CPATC databases were used to analyze the mRNA and protein levels of MMS21 in adjacent and carcinoma tissues;the endogenous expression levels of MMS21 were reduced individually by two siRNAs.Meanwhile,flow cytometry was used to detect the distribution ratio of cell cycle of hepatocellular carcinoma cells.BrdU staining and DNA fiber assay were employed to determine cells proliferation rate,and colony assay was used to evaluate the ability of cell proliferation;the MYC tagged MMS21 was cloned and transfected into SK-hep1 to generate MMS21 stably overex⁃pressed cells.Co-IP combined with mass spectrometry were used to identify MMS21-interacting proteome and map interac⁃tive pathway;Western Blot(WB)was performed to determine the effect of MMS21 towards cell cycle-related proteins.【Results】Compared with adjacent tissues(n=160),MMS21 was significantly higher expressed in cancerous tissues(n=369;P<0.05)and its high expression was associated with poor prognosis(P<0.01)in hepatocellular carcinoma;Knockdown of endogenous MMS21 by two siRNAs led to the increase of the proportion of cells in G0/G1 phase(P<0.001;P<0.01),and the decrease of the ratio of cells in S(P<0.01;P<0.05),G2/M(P<0.05;P<0.05)phase.Moreover,depletion of endogenous MMS21 greatly attenuated the speed of DNA replication(P<0.0001;P<0.0001)and the rate of cell proliferation(P<0.01;P<0.001).Mass spectrometry analysis identified the total of 641 high-confidence proteins for MMS21,and interactive pathway analysis showed that these binding proteins were highly correlated with cell cycle-re⁃lated genes.The results of Western Blot further confirmed this conclusion,and depletion of endogenous MMS21 markedly reduced the protein levels of Microchromosome maintenance protein family(MCMs).【Conclusions】MMS21 is highly ex⁃pressed in hepatocellular carcinoma and positively correlated with the poor prognosis.MMS21 is closely rela

关 键 词：肝癌 MMS21 增殖 CoIP-MS 微小染色体维持蛋白家族 

分 类 号：R735.7[医药卫生—肿瘤]