黏蛋白1调控肿瘤细胞恶性特征的功能位点分析  

Functional site analysis of mucin 1 in regulating the malignant characteristics of tumor cells

作　　者：高珂星 廖春华 李昇泽 马双羽 黄雷[1] GAO Kexing;LIAO Chunhua;LI Shengze;MA Shuangyu;HUANG Lei(Department of Histoembryology,Genetics and Developmental Biology,Shanghai Jiao Tong University College of Basic Medical Sciences,Shanghai 200025,China)

机构地区：[1]上海交通大学基础医学院组织胚胎学与遗传发育学系,上海200025

出　　处：《上海交通大学学报（医学版）》2024年第11期1370-1382,共13页Journal of Shanghai Jiao tong University:Medical Science

基　　金：国家自然科学基金(81874197,82073111);上海市科学技术委员会“科技创新行动计划”生物医药科技支撑专项(21S11901600)。

摘　　要：目的·探究黏蛋白1(mucin 1,MUC1)调控肿瘤细胞增殖、迁移和干性维持的功能位点。方法·通过癌症基因组图谱(The Cancer Genome Atlas,TCGA)数据库分析寻找MUC1基因在不同癌症中的突变特征,对不同MUC1突变位点进行分析及定位,并按突变出现频率排序;通过Western blotting筛选出突变频率较高且蛋白稳定表达的MUC1突变体,利用乳腺癌细胞株BT549敲除MUC1细胞系和乳腺非转化细胞株MCF-10A,应用慢病毒表达系统构建MUC1野生型(MUC1-WT)和突变体稳定表达的细胞系。采用免疫荧光法检测不同MUC1突变体的细胞定位。以MUC1-WT为阳性对照、MUC1-AQA功能丧失突变体为阴性对照,对不同突变细胞的肿瘤生物学功能进行分析:通过细胞计数试剂盒-8(cell counting kit-8,CCK-8)及克隆形成实验检测细胞增殖能力;通过划痕实验及Transwell实验检测细胞迁移能力;通过成球实验检测细胞干性。使用PyMOL软件分析MUC1突变体结构定位并通过蛋白质对接软件(ZDOCK Server)进行分子对接分析。结果·在TCGA数据库中得到102个位于MUC1编码区的突变,其中P418S、S251R、V359I、N271S、N465H 5个错义突变出现频率较高且位于非数目可变串联重复序列(non-variable number of tandem repeats,non-VNTR)区域。进一步检测发现MUC1-S251R、N271S、V359I突变体可稳定表达;细胞定位分析发现这3个突变体主要分布于细胞质,同时细胞核也有一定的分布,核质比与野生型未见明显差异。表达不同MUC1突变体细胞的肿瘤生物学功能分析发现:①MUC1-WT高表达显著增强BT549和MCF-10A细胞的增殖能力;与MUC1-WT细胞相比,MUC1-AQA、S251R、N271S突变体细胞增殖能力下降,但MUC1-V359I突变体细胞与MUC1-WT细胞具有相似的增殖能力。②MUC1-WT高表达细胞的迁移能力显著增强,而MUC1-AQA细胞迁移能力减弱。在BT549细胞中,MUC1-S251R与MUC1-V359I突变体细胞迁移能力与MUC1-WT细胞相似,但MUC1-N271S细胞的Objective·To identify the functional motifs of mucin 1(MUC1)involved in regulating tumor cell proliferation,migration,and stemness maintenance.Methods·Mutational characteristics of the MUC1 gene across different cancers were identified from The Cancer Genome Atlas(TCGA)database.Various MUC1 mutation sites were analyzed and localized,followed by ranking based on mutation frequency.Western blotting was used to screen high-frequency MUC1 mutants with stable protein expression.BT549 cell line with MUC1 knocked out and MCF-10A cell line were used to stably overexpress MUC1 wild-type(MUC1-WT)and mutants by using lentiviral technology.Immunofluorescence was used to detect the cellular localization of MUC1 mutants.Using MUC1-WT as a positive control and MUC1-AQA,a loss-of-function mutant,as a negative control,the biological functions of different MUC1 mutant cells were analyzed:cell proliferation ability was assessed by cell counting kit-8(CCK-8)assay and colony formation assay;cell migration ability was evaluated by wound-healing and Transwell assays;cell stemness was examined by sphere formation assay.Structural localization of MUC1 mutants was analyzed by using PyMOL software,and molecular docking analysis was performed by using a protein docking software(ZDOCK Server).Results·A total of 102 mutations located in the MUC1 coding region were identified in the TCGA database,among which five missense mutations(P418S,S251R,V359I,N271S,and N465H)exhibited higher frequencies and were located in the non-variable number of tandem repeats(non-VNTR)region.Further examination revealed that the MUC1-S251R,N271S,and V359I mutants could be stably expressed.The cellular localization assay indicated that these three mutants predominantly localized in the cytoplasm,but were also presented in the nucleus.The nuclear-to-cytoplasmic ratio showed minimal differences between MUC1-WT and the mutants.Analysis of the tumorigenic biological functions of the cells expressing different MUC1 mutants revealed that:①High expression of MUC1-WT s

关 键 词：黏蛋白1 错义突变 肿瘤 细胞增殖 细胞迁移 细胞干性 

分 类 号：R730.2[医药卫生—肿瘤]