NKX2-5在乳腺癌患者他莫昔芬耐药中的作用及调控机制  

Role of NKX2-5 on tamoxifen resistance in breast cancer patients and its regulation mechanism

作　　者：赵铮 李毅 杨飞翔 ZHAO Zheng;LI Yi;YANG Feixiang(Department of Clinical Laboratory,Dongfeng Hospital,Hubei University of Medicine,Shiyan 442000,Hubei,China)

机构地区：[1]湖北医药学院附属东风医院检验科

出　　处：《检验医学》2020年第1期47-55,共9页Laboratory Medicine

摘　　要：目的探讨NKX2-5在乳腺癌患者他莫昔芬(TAM)耐药中的作用及调控机制。方法采用免疫组化染色检测乳腺癌组织和癌旁组织NKX2-5的表达。利用高通量基因表达数据库(GEO)分析乳腺癌患者的相关资料及NKX2-5表达与乳腺癌患者生存率的关系。构建乳腺癌TAM耐药细胞株(MCF-7/TAM),采用CCK-8试剂盒检测不同浓度TAM处理后细胞增殖的变化,免疫印迹法检测细胞相关蛋白表达,逆转录-聚合酶链反应(RT-PCR)检测细胞NKX2-5 mRNA的表达。结果癌旁组织NKX2-5表达明显高于癌组织(P<0.05)。TAM敏感组织NKX2-5表达明显高于TAM耐药组织(P<0.05)。NKX2-5低表达组总生存率和无病生存率明显低于NKX2-5高表达组(P<0.05);在接受TAM治疗的乳腺癌患者中,NKX2-5低表达组总生存率明显低于NKX2-5高表达组(P<0.05)。与MCF-7细胞比较,MCF-7/TAM细胞NKX2-5蛋白表达明显降低(P<0.05)。采用瞬时转染技术将NKX2-5过表达质粒(Ad-NKX2-5)、空载质粒(Ad-NC)转染MCF-7/TAM细胞(Ad-NKX2-5组、Ad-NC组),NKX2-5小干扰RNA质粒(NKX2-5-siRNA)、空白对照(NC-siRNA)转染MCF-7细胞(NKX2-5-siRNA组、NC-siRNA组)。采用50、100μmol/L TAM处理后,Ad-NKX2-5组细胞增殖抑制率明显高于Ad-NC组(P<0.05),NKX2-5-siRNA组细胞增殖抑制率明显低于NC-siRNA组(P<0.05)。MCF-7/TAM细胞磷酸化p38丝裂原活化蛋白激酶(p-P38MAPK)、磷酸化c-Jun氨基末端激酶(p-JNK)、磷酸化细胞外信号调节激酶(p-ERK)蛋白表达明显高于MCF-7细胞(P<0.05)。Ad-NKX2-5组p-P38、p-JNK、p-ERK蛋白表达明显低于Ad-NC组(P<0.05),NKX2-5-siRNA组p-P38、p-JNK、p-ERK蛋白表达明显高于NC-siRNA组(P<0.05)。结论乳腺癌TAM耐药细胞中NKX2-5表达下调。上调NKX2-5表达或许能通过抑制丝裂原活化蛋白激酶(MAPK)信号通路逆转MCF-7细胞TAM耐药。Objective To investigate the role of NKX2-5 on tamoxifen(TAM)resistance in breast cancer and its regulation mechanism.Methods The expressions of NKX2-5 in breast cancer tissues and adjacent tissues were determined by immunohistochemical staining assay.Using Gene Expression Omnibus(GEO)database,the relative data of breast cancer patients were analyzed,and the correlation between NKX2-5 and the survival rate of breast cancer patients was analyzed.The TAM-resistant cell lines(MCF-7/TAM)were constructed,and cell viability was determined by CCK-8 kit after the treatment with different concentrations of TAM.The expressions of the related proteins were determined by western blotting,and NKX2-5 mRNA expression was determined by reverse transcription polymerase chain reaction(RT-PCR).Results The expression of NKX2-5 was higher in adjacent tissues than that in cancer tissues(P<0.05),and NKX2-5 expression in TAM-sensitive tissues was higher than that in TAM-resistant tissues(P<0.05).Low expression of NKX2-5 was associated with poor overall survival rate and relapse free survival rate in breast cancer patients(P<0.05),and high NKX2-5 expression level also predicted better outcome for patients with TAM endocrine therapy(P<0.05).Compared to the MCF-7 cells,the expression of NKX2-5 protein was decreased in MCF-7/TAM cells(P<0.05).Overexpression NKX2-5 plasmid(Ad-NKX2-5)and empty plasmid (Ad-NC) were transfected in MCF-7/TAM cell(Ad-NKX2-5 group and Ad-NC group) by transient transfectiontechnology. NKX2-5-small interfering RNA plasmid(NKX2-5-siRNA) and blank control(NC-siRNA) weretransfected in MCF-7 cell(NKX2-5-siRNA group and NC-siRNA group). After treatment with 50 and 100 μmol/LTAM,the cell proliferation inhibition rates were increased in Ad-NKX2-5 group compared to Ad-NC group(P<0.05).The cell proliferation inhibition rates were decreased in NKX2-5-siRNA group compared to NC-siRNA group(P<0.05).The expressions of phosphorylated-P38 mitogen-activated protein kinase(p-P38MAPK),phosphorylated-c-JunN-terminal kinase(p-JNK),pho

关 键 词：转录因子NKX2-5 乳腺癌 他莫昔芬 丝裂原活化蛋白激酶信号通路 

分 类 号：R446.1[医药卫生—诊断学]