低氧相关的R-脊椎蛋白3对人乳腺导管癌细胞增殖能力的影响及其机制  

Effect of hypoxia-related R-spondin3 on proliferation of human breast ductal carcinoma cells and underlying mechanism

作　　者：马德寿[1] 蒋树云 张英 王宁 马志军 Ma Deshou;Jiang Shuyun;Zhang Ying;Wang Ning;Ma Zhijun(Department of Surgical Oncology,Affiliated Hospital of Qinghai University,Xining 810001,China;Postgraduate School of Qinghai University,Xining 810006,China)

机构地区：[1]青海大学附属医院肿瘤外科,西宁8810001 [2]青海大学研究生院,西宁810016

出　　处：《中华实验外科杂志》2023年第4期639-641,共3页Chinese Journal of Experimental Surgery

基　　金：青海省科技厅应用基础研究项目(2019-ZJ-7091)。

摘　　要：目的体外观察低氧微环境对乳腺癌细胞增殖能力的影响,并探讨其分子机制。方法细胞增殖检测试剂盒(CCK-8)用于人乳腺导管癌细胞株(BT-474,购于中国科学院上海细胞库)经低氧处理后的增殖能力;应用基因芯片对低/常氧处理后的细胞进行差异基因的表达检测,并在体外行实时定量聚合酶链反应(RT-PCR)验证;运用癌症基因组图谱(TCGA)及人类蛋白质表达图谱(HPA)数据库分析筛选的差异基因在乳癌及正常乳腺组织中的表达特征,并分析其水平与患者临床病理学特征及预后的相关性;基因组百科全书(KEGG)通路分析、基因过表达实验、蛋白质印迹法(Western blot)等方法探讨其发挥功能的分子机制,组间比较采用t检验。结果BT-474细胞分别经常氧、低氧干预24、36、48 h后,常氧组中的吸光度值显著低于低氧组(0.32±0.03比0.40±0.02,t=6.351;0.45±0.01比0.52±0.02,t=4.196;0.57±0.04比0.68±0.03,t=3.257;P<0.05);基因芯片分析结果提示R-脊椎蛋白3(r-spondin3,RSPO3)是细胞经低氧处理后显著抑制表达的基因之一;进一步的RT-PCR结果示,低氧组中RSPO3的相对表达量显著低于常氧组(0.56±0.03比0.82±0.04,t=12.583,P<0.05);TCGA分析结果示RSPO3在乳腺癌组织中的表达量较乳腺正常组织显著降低(1.41±0.37比3.54±0.19,t=14.648,P<0.01),其在乳腺癌组织中表达水平与肿瘤的直径呈负相关(r=-0.693,P<0.01);生存分析结果表明,RSPO3低表达组中患者的总生存期(OS)显著低于高表达组[(46.52±0.28)个月比(53.79±0.47)个月,t=21.357,P<0.01];此外,KEGG分析结果表明,细胞经低氧处理后β-catenin通路被预测为显著激活;Western blot结果示低氧组细胞核内β-catenin的表达量较常氧组显著升高(0.89±0.06比0.42±0.02,t=6.241,P<0.05);与低氧组比较,低氧下过表达RSPO3后24、36、48 h后细胞的吸光度值显著降低(0.43±0.03比0.32±0.02,t=4.458;0.51±0.01比0.39±0.02,t=3.267;0.66±0.03比0.49±0.04,t=4.103;P<0Objective To observe the effect of hypoxia on the proliferation of breast cancer cells,and explore its related mechanism.Methods Cell counting kit(CCK-8)assay was used to detect the proliferation ability of human breast ductal carcinoma cells(BT-474)under hypoxia.The gene chip was used to detect differentially expressed genes after hypoxia.Teverse transcription-polymerase chain reaction(RT-PCR)was used to examine the levels of R-spondin3(RSPO3)in BT-474 cells after hypoxia,then the cancer genome atlas(TCGA)and human protein atls(HPA)database were used to analyze the correlation between RSPO3 expression and the clinical pathological characteristics as well as prognosis.Kyoto Encyclopedia of Genes and Genomes(KEGG),gene overexpression in vitro and Western blotting were used to explore the molecular mechanism.Differences between groups were compared using t test.Results After 24,36 and 48 h,the absorbance of BT-474 cells in the normoxic group was significantly lower than that in the hypoxic group(0.32±0.03 vs.0.40±0.02,t=6.351;0.45±0.01 vs.0.52±0.02,t=4.196;0.57±0.04 vs.0.68±0.03,t=3.257,all P<0.05).RSPO3 was down-regulated under hypoxia.The RT-PCR results showed that the level of RSPO3 in hypoxia group was significantly lower than that in normoxia group(0.56±0.03 vs.0.82±0.04,t=12.583,P<0.05).TCGA analysis showed that the expression of RSPO3 in breast cancer tissue was significantly lower than that in normal breast tissue(1.41±0.37 vs.3.54±0.19,t=14.648,P<0.01),and its expression was negatively correlated with diameter of the tumor(r=-0.693,P<0.01).Survival analysis indicated that the overall survival(OS)of patients in the low expression group of RSPO3 was significantly shorter than that in the high expression group[(46.52±0.28)months vs.(53.79±0.47)months,t=21.357,P<0.01].In addition,KEGG analysis showed that theβ-catenin pathway was predicted to be significantly activated under hypoxia.Western blotting results showed the expression ofβ-catenin in the hypoxic group was significantly higher than the n

关 键 词：乳腺癌 低氧 R-脊椎蛋白3 增殖 

分 类 号：R737.9[医药卫生—肿瘤]