NKX2-5在乳腺癌患者他莫昔芬耐药中的作用及调控机制

doi:10.3969/j.issn.1673-8640.2020.01.011

摘要/Abstract

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、100 μmol/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。

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

Abstract:

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 transfection technology. NKX2-5-small interfering RNA plasmid（NKX2-5-siRNA） and blank control（NC-siRNA） were transfected in MCF-7 cell（NKX2-5-siRNA group and NC-siRNA group）. After treatment with 50 and 100 μmol/L TAM,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-Jun N-terminal kinase（p-JNK）,phosphorylated-extracellular regulated protein kinase（p-ERK）proteins were increased in MCF-7/TAM cell compared to MCF-7 cell（P<0.05）. The expressions of p-P38,p-JNK and p-ERK proteins were increased in Ad-NKX2-5 group compared to Ad-NC group（P<0.05）. Meanwhile,the expression of p-P38,p-JNK and p-ERK proteins were decreased in NKX2-5-siRNA group compared to NC-siRNA group（P<0.05）. Conclusions NKX2-5 is down-regulated in TAM-resistant breast cancer cell,and overexpressive NKX2-5 overcomes TAM resistance in breast cancer via suppressing mitogen-activated protein kinase（MAPK） signaling pathway.

Key words: Transcriptional factor NKX2-5, Breast cancer, Tamoxifen, Mitogen-activated protein kinase signaling pathway

中图分类号:

R446.1

赵铮, 李毅, 杨飞翔. NKX2-5在乳腺癌患者他莫昔芬耐药中的作用及调控机制[J]. 检验医学, 2020, 35(1): 47-55.

ZHAO Zheng, LI Yi, YANG Feixiang. Role of NKX2-5 on tamoxifen resistance in breast cancer patients and its regulation mechanism[J]. Laboratory Medicine, 2020, 35(1): 47-55.

图/表 12

图1 不同组织中NKX2-5的表达（×200）注:（a）乳腺癌组织;（b）癌旁组织;（c）TAM耐药的乳腺癌组织;（d）TAM敏感的乳腺癌组织

表1 乳腺癌患者的一般资料

患者来源	例数	年龄/岁	TNM分期				ER表达			PR表达			HER-2表达
患者来源	例数	年龄/岁	Ⅰ期	Ⅱ期	Ⅲ期	Ⅳ期	阳性	阴性	不详	阳性	阴性	不详	阳性	阴性	不详
本研究	50	44.5±9.4	8	23	17	2	37	13	0	32	18	0	21	29	0
GEO	104	45.1±8.7	18	52	27	7	67	30	7	60	24	20	30	62	12

图2 不同组织免疫组化染色评分比较注:（a）乳腺癌组织与癌旁组织免疫组化染色评分的比较;（b）TAM敏感组织与TAM耐药组织免疫组化染色评分的比较

图3 NKX2-5高表达和低表达乳腺癌患者的Kaplan-Meier生存曲线注:（a）总体生存率;（b）无复发生存率;（c）接受TAM治疗的乳腺癌患者的生存率;（d）未接受TAM治疗的乳腺癌患者的生存率; NKX2-5高表达 ;NKX2-5低表达

图4 MCF-7、T47D、BCAP37、SKBR3及MCF10A细胞NKX2-5蛋白及NKX2-5 mRNA的表达注:（a）NKX2-5蛋白表达的免疫印迹图;（b）NKX2-5蛋白的相对表达量;与MCF10A细胞比较,*P<0.05;（c）NKX2-5 mRNA的相对表达量;与MCF10A细胞比较,*P<0.05

表2 不同浓度TAM对MCF-7、T47D、BCAP37、SKBR3细胞的增殖抑制率 %,$\bar{x}$±s

细胞株	TAM/（μmol/L）
细胞株	5	20	50	100
MCF-7	26.3±3.7	89.4±5.8	91.4±6.7	82.4±6.5
T47D	28.2±2.5	47.8±7.1	63.5±10.4	63.4±5.3
BCAP37	21.7±4.3	31.2±4.7	68.1±6.4	60.7±5.2
SKBR3	25.2±3.1	38.5±7.3	58.6±5.4	54.6±6.6
MCF-7/TAM	24.5±3.7	31.6±5.3	53.1±6.1	50.4±4.8

图5 MCF-7和MCF-7/TAM细胞NKX2-5蛋白表达的比较注:（a）NKX2-5蛋白表达的免疫印迹图;（b）NKX2-5蛋白的相对表达量

图6 不同浓度TAM处理后MCF-7和MCF-7/TAM细胞NKX2-5蛋白表达的比较注:（a）NKX2-5蛋白表达的免疫印迹图;（b）NKX2-5蛋白的相对表达量;0 μmol/L TAM;5 μmol/L TAM;10 μmol/L TAM

图7 Ad-NKX2-5组、Ad-NC组、NKX2-5-siRNA组和NC-siRNA组NKX2-5蛋白的表达注:（a）Ad-NKX2-5组和Ad-NC组NKX2-5蛋白表达的免疫印迹图;（b）Ad-NKX2-5组和Ad-NC组NKX2-5蛋白的相对表达量;（c）NKX2-5-siRNA组和NC-siRNA组NKX2-5蛋白表达的免疫印迹图;（d）NKX2-5-siRNA组和NC-siRNA组NKX2-5蛋白的相对表达量

表3 不同浓度TAM对Ad-NKX2-5组、Ad-NC组和NKX2-5-siRNA组、NC-siRNA组的增殖抑制率 %,$\bar{x}$±s

组别	TAM/（μmol/L）
组别	5	20	50	100
Ad-NKX2-5组	8.7±0.5	21.4±5.7	56.9±10.1^*	67.9±7.8^*
Ad-NC组	4.3±0.4	19.7±4.5	28.9±7.3	49.7±7.1
NKX2-5- siRNA组	19.8±5.4	38.9±6.2^*#	40.9±5.4^*#	40.5±5.6^*#
NC-siRNA组	21.2±6.5	59.4±8.4	60.1±8.9	67.7±7.6

图8 MCF-7细胞和MCF-7/TAM细胞MAPK信号通路相关蛋白的表达注:（a）各蛋白表达的免疫印迹图;（b）各蛋白的相对表达量;MCF-7细胞;MCF-7/TAM细胞

图9 Ad-NKX2-5组、Ad-NC组、NKX2-5-siRNA组和NC-siRNA组MAPK信号通路相关蛋白的表达注:（a）Ad-NKX2-5组和Ad-NC组各蛋白表达的免疫印迹图;（b）Ad-NKX2-5组和Ad-NC组各蛋白的相对表达量; Ad-NKX2-5组;Ad-NC组;（c）NKX2-5-siRNA组和NC-siRNA组各蛋白表达的免疫印迹图;（d）NKX2-5-siRNA组和NC-siRNA组各蛋白的相对表达量;NKX2-5-siRNA组;NC-siRNA组

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