G蛋白核仁3在乳腺癌中的表达及其对血管生成的影响和机制  

Expression of G protein nucleolar 3in breast cancer and its effect on angiogenesis and mechanism

作　　者：马晓燕 黄冰 张文超 张稳 黄桂林[1] 孙旭凌[1] MA Xiaoyan;HUANG Bing;ZHANG Wenchao;ZHANG Wen;HUANG Guilin;SUN Xuling(First Affiliated Hospital,Shihezi University,Shihezi,Xinjiang832008,China;School of Medicine,Shihezi University,Shihezi,Xinjiang832002,China)

机构地区：[1]石河子大学第一附属医院胃肠外科,新疆石河子832008 [2]石河子大学第一附属医院心胸外科,新疆石河子832008 [3]石河子大学医学院,新疆石河子832002

出　　处：《中华肿瘤防治杂志》2024年第24期1504-1517,共14页Chinese Journal of Cancer Prevention and Treatment

基　　金：国家自然科学基金(82260521);石河子大学创新发展专项基金(CXFZ202113);新疆生产建设兵团博士后基金(308090);北京科创医学发展基金会项目(KC2023-JX-0186-FQ037);天山英才科技创新团队:中亚地区高发疾病防治应用研究创新团队(2023TSYCTD0020)。

摘　　要：目的检测G蛋白核仁3(GNL3)在乳腺癌中的表达及预后价值,探讨GNL3在乳腺癌血管生成中的作用机制。方法通过数据库和免疫组织化学(IHC)分析乳腺癌组织和正常组织GNL3的表达情况及其与乳腺癌预后的关系。用敲低和高表达GNL3的慢病毒分别转染MDA-MB-231和MCF-7细胞。分为GNL3敲低慢病毒(shGNL3#1、shGNL3#2)组及其空转病毒(shNC)组、GNL3高表达慢病毒(GNL3)组及其空转病毒(Vector)组。使用稳定敲低或高表达GNL3的乳腺癌细胞系制备的条件培养基孵育人脐静脉内皮细胞(HUVECs)。采用细胞计数试剂盒8(CCK8)实验、流式细胞术、内皮细胞成管能力检测实验分析GNL3对HUVECs细胞生长及成管能力的影响。通过基因富集分析(GESA)、实时荧光定量聚合酶链式反应(qRT-PCR)、酶联免疫吸附实验(ELISA)和蛋白质印迹法分析GNL3和血管内皮生长因子-A(VEGFA)的关系,通过动物实验进一步验证这一关系,并通过免疫共沉淀(IP)及染色质免疫共沉淀(ChIP)实验来探讨其内在调控机制。采用独立样本t检验、单因素方差分析和χ^(2)检验比较组间差异。结果数据库分析结果显示,在GSE15852和GSE109169数据集中,GNL3在乳腺癌组织中的表达水平均高于正常组织,t值分别为-2.488和-2.201,P值分别为0.015和0.036。生存分析结果显示,GSE20711数据集中GNL3高表达与更差的总生存期(OS;HR=3.56,95%CI:1.42~8.92,P=0.004)和无复发生存期(RFS;HR=1.96,95%CI:1.03~3.75,P=0.037)有关。CCK8实验结果显示,乏氧条件下与shNC组(0.659±0.012)相比,shGNL3#1组(0.521±0.010)和shGNL3#2组(0.554±0.019)HUVECs细胞增殖能力降低,t值分别为15.478和8.080,均P<0.001。流式细胞术检测结果显示,在乏氧条件下,与shNC组相比,shGNL3#1组和shGNL3#2组HUVECs细胞处于G2/M期比例增多,差异有统计学意义,χ^(2)=110.165,P<0.001。内皮细胞成管能力检测结果显示,在乏氧条件下,shNC组HUVECs细胞成管能力为35.000±5.000,shGNLObjective To detect the expression of G protein nucleolus 3(GNL3)in breast cancer and its prognostic value,and to explore the mechanism of GNL3in angiogenesis of breast cancer.Methods The expression of GNL3in breast cancer tissues and normal tissues and its relationship with the prognosis of breast cancer were analyzed by database and immunohistochemistry(IHC).Transfect MDA-MB-231and MCF-7cells with knockdown and high expression of GNL3 lentivirus,respectively.Divided into GNL3knockdown lentivirus(shGNL3#1,shGNL3#2)group and its idle virus(shNC)group,GNL3high expression lentivirus(GNL3)group and its idle virus(Vector)group.Human umbilical vein endothelial cells(HUVECs)were incubated in conditioned medium prepared by breast cancer cell lines stably knockdown or overexpression of GNL3.The effects of GNL3on the growth and tube forming ability of HUVECs were analyzed by using Cell Counting Kit 8(CCK8)assay,flow cytometry,and endothelial cell tube forming ability assay.The relationship between GNL3and vascular endothelial growth factor-A(VEGFA)was analyzed through gene enrichment analysis(GESA),real-time fluorescence quantitative polymerase chain reaction(qRT-PCR),enzyme-linked immunosorbent assay(ELISA),and Western blotting.This relationship was further validated through animal experiments,and its underlying regulatory mechanism was explored through immunoprecipitation(IP)and chromatin immunoprecipitation(ChIP)experiments.Independent sample t-test,one-way ANOVA,andχ^(2)-test were used to compare the differences between groups.Results The results of database analysis showed that in GSE15852and GSE109169data sets,the expression level of GNL3in breast cancer tissues was higher than that in normal tissues,with t values of-2.488and-2.201,respectively,and P values of 0.015and 0.036.The survival analysis results showed that high expression of GNL3in the GSE20711dataset was associated with poorer overall survival(OS;HR=3.56,95%CI:1.42-8.92,P=0.004)and recurrence free survival(RFS;HR=1.96,95%CI:1.03-3.75,P=0.037).The CCK8exp

关 键 词：G蛋白核仁3 血管内皮生长因子-A 血管生成 乳腺癌 缺氧诱导因子-1Α 

分 类 号：R737.9[医药卫生—肿瘤]