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三阴性乳腺癌(triple-negative breast cancer, TNBC)是一种缺乏人类表皮生长因子受体2和雌激素、孕激素表达的乳腺癌亚型[1]。TNBC组织学上分化低、增殖快、转移早、临床上抗激素和HER2治疗无效,目前TNBC的治疗主要行常规化疗,循证医学[2]表明TNBC病人复发率高、预后差、生存时间短。因此,发现靶基因免疫治疗对TNBC的临床诊断、治疗和预后有重要意义[3]。有研究[4]表明组成型激活信号转导激活剂3(STAT3)可参与TNBC的血管生成、发生发展、侵袭和迁移。短发夹RNA(short hairpin RNA, shRNA)是一种DNA分子,可以克隆到表达载体中,表达小干扰RNA(small interfering RNA, siRNA, RNA双链19~21个核苷酸)[5]。基于STAT3设计shRNA序列并将其克隆成特定的载体,本文旨在探讨STAT3在TNBC中的调控机制及其作为治疗靶点的应用前景。
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STAT3蛋白主要在细胞核中表达。结果表明,正常乳腺组织核染色表达减弱甚至缺失,而乳腺癌组织核染色表达呈阳性, 组间差异有统计学意义(χ2=8.53, P < 0.01)(见图 1、表 1)。
正常乳腺组织 乳腺癌组织 合计 + - + 21 13 34 - 82 14 96 合计 103 27 130 表 1 STAT3在不同乳腺组织中的表达
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TNBC中STAT3蛋白的高表达与肿瘤大小、组织学分级、TNM分期和淋巴结转移呈正相关(P < 0.05~P < 0.01)。即肿瘤体积越大,组织学分级越高,TNM分期越晚,伴有淋巴结转移的乳腺癌中STAT3阳性表达率越高(见表 2)。
因素 STAT3 zc P - + 肿瘤大小 ≤2 cm 5 45 > 2~5 cm 8 26 2.47 < 0.05 > 5 cm 14 32 组织学分级 Ⅰ 4 45 Ⅱ 8 23 2.66 < 0.01 Ⅲ 15 35 TNM分期 Ⅰ 2 39 Ⅱ
Ⅲ5
730
234.46 < 0.01 Ⅳ 13 11 淋巴结转移 0 1 36 1~3
4~93
721
334.91 < 0.01 > 9 16 13 表 2 STAT3表达与乳腺癌临床病理特征的联系
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乳腺癌组织中STAT3蛋白的表达(1.01±0.02)显著高于正常乳腺组织(0.24±0.02)(t=-310.40,P < 0.01)(见图 2)。MDA-MB-231转染48 h后,实验组STAT3蛋白与阴性对照组和空白对照组相比表达降低,差异有统计学意义(P < 0.01)(见图 3、表 3)。
分组 n STAT3蛋白相对表达量 STAT3 mRNA相对表达量 空白对照组 130 1.08±0.02 1.25±0.04 阴性对照组 130 0.96±0.01** 1.15±0.02** 实验组 130 0.27±0.05**△△ 0.31±0.03**△△ F — 24 843.00 35 844.14 P — < 0.01 < 0.01 MS组内 — 0.001 0.001 q检验:与空白对照组比较** P < 0.01;与阴性对照组比较△△ P < 0.01 表 3 shRNA转染后MDA-MB-231细胞中STAT3的蛋白和mRNA表达比较(x ± s)
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MDA-MB-231转染48 h后,实验组mRNA表达水平与阴性对照组和空白对照组相比表达降低,差异有统计学意义(P < 0.01)(见表 3)。
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CCK-8检测结果显示,MDA-MB-231转染24、48和72 h后,细胞生长抑制率分别为5.6%、17.2%和39.1%。实验组细胞增殖活性与阴性对照组和空白对照组相比下降缓慢,差异有统计学意义(P < 0.01)(见表 4)。
分组 n 细胞增殖活性 空白对照组 130 1.15±0.02 阴性对照组 130 1.23±0.03** 实验组 130 0.36±0.01**△△ F — 64 415.00 P — < 0.01 MS组内 — 0.001 q检验:与空白对照组比较** P < 0.01;与阴性对照组比较△△ P < 0.01 表 4 STAT3-shRNA对MDA-MB-231增殖率的影响(x ± s)
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Transwell实验表明,实验组细胞的侵袭和迁移能力低于阴性对照组和空白对照组(见表 5)。侵袭和迁移实验结果显示,实验组通过基底膜的细胞数与阴性对照组和空白对照组相比显著减少,差异有统计学意义(P < 0.01)(见图 4)。
分组 n 穿膜细胞数量(侵袭) 穿膜细胞数量(迁移) 空白对照组 130 186.0±5.4 115.6±4.3 阴性对照组 130 158.8±6.5** 117.9±3.6** 实验组 130 36.4±6.2**△△ 32.5±2.3**△△ F — 22 545.42 25 129.67 P — < 0.01 < 0.01 MS组内 — 36.617 12.246 q检验:与空白对照组比较** P < 0.01;与阴性对照组比较△△ P < 0.01 表 5 STAT3对MDA-MB-231侵袭和迁移能力的影响(x ± s)
STAT3蛋白在三阴性乳腺癌中的作用和临床病理意义
Role and clinicopathological significance of STAT3 protein in triple-negative breast cancer
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摘要:
目的探讨组成型激活信号转导激活剂3(STAT3)在三阴性乳腺癌(TNBC)组织中的表达,分析其作为TNBC病人靶向治疗的分子标志物的意义,探究TNBC的发病机制、指导治疗和临床预后。 方法免疫组织化学方法检测STAT3基因在TNBC中的表达并解析其与临床病理的关系。构建STAT3-shRNA表达载体建立下调STAT3基因表达的TNBC细胞系(MDA-MB-231)。CCK-8和Transwell实验验证转染前后MDA-MB-231增殖、侵袭和迁移能力的差异。 结果STAT3蛋白在TNBC中的表达水平显著高于正常乳腺(P < 0.01),且在TNBC中STAT3阳性表达率与肿瘤体积、组织学分级、TNM分期、淋巴结转移呈正相关(P < 0.05~P < 0.01)。MDA-MB-231细胞转染后实验组STAT3蛋白表达与阴性对照组和空白对照组相比显著降低(P < 0.01)。实验组细胞增殖活性与阴性对照组和空白对照组相比缓慢下降(P < 0.01)。实验组细胞穿过基底膜的数量与阴性对照组和空白对照组比较显著减少(P < 0.01)。 结论STAT3-shRNA表达载体通过RNA干扰有效下调STAT3基因在TNBC中的表达。靶向STAT3-shRNA可抑制MDA-MB-231细胞的增殖、侵袭和迁移及促凋亡能力。 -
关键词:
- 三阴性乳腺癌 /
- 组成型激活信号转导激活剂3 /
- 短发夹RNA /
- 基因调控
Abstract:ObjectiveTo investigate the expression of STAT3 in triple-negative breast cancer(TNBC) tissue, analyze its significance as a molecular marker for targeted therapy to TNBC patients, and explore the pathogenesis, clinical efficacy and prognosis of TNBC. MethodsThe expression of STAT3 gene in TNBC was detected by immunohistochemistry, and its relationship with clinicopathological features was analyzed.The STAT3-shRNA expression vector was constructed to establish a TNBC cell line(MDA-MB-231) with down-regulated STAT3 gene expression.The differences in cell proliferation, invasion and migration abilities before and after transfection were analyzed by CCK-8 and Transwell assay. ResultsThe expression of STAT3 protein in TNBC tissue was significantly higher than that in breast tissue(P < 0.01), and the positive expression rate of STAT3 in TNBC was positively correlated with tumor volume, histological grade, TNM stage and lymph node metastasis(P < 0.05 to P < 0.01).Compared with the negative control group and blank control group, the expression of STAT3 protein in the experimental group was significantly decreased after transfection in MDA-MB-231 cells, the cell proliferation activity in the experimental group decreased slowly, and the number of cells passing through the basement membrane in the experimental group was significantly reduced(P < 0.01). ConclusionsThe STAT3-shRNA expression vector effectively down-regulates the expression of STAT3 gene in TNBC by RNA interference.Targeting STAT3-shRNA can inhibit the proliferation, invasion and migration and pro-apoptotic ability of MDA-MB-231 cells. -
Key words:
- triple-negative breast cancer /
- STAT3 /
- short hairpin RNA /
- gene regulation
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表 1 STAT3在不同乳腺组织中的表达
正常乳腺组织 乳腺癌组织 合计 + - + 21 13 34 - 82 14 96 合计 103 27 130 表 2 STAT3表达与乳腺癌临床病理特征的联系
因素 STAT3 zc P - + 肿瘤大小 ≤2 cm 5 45 > 2~5 cm 8 26 2.47 < 0.05 > 5 cm 14 32 组织学分级 Ⅰ 4 45 Ⅱ 8 23 2.66 < 0.01 Ⅲ 15 35 TNM分期 Ⅰ 2 39 Ⅱ
Ⅲ5
730
234.46 < 0.01 Ⅳ 13 11 淋巴结转移 0 1 36 1~3
4~93
721
334.91 < 0.01 > 9 16 13 表 3 shRNA转染后MDA-MB-231细胞中STAT3的蛋白和mRNA表达比较(x ± s)
分组 n STAT3蛋白相对表达量 STAT3 mRNA相对表达量 空白对照组 130 1.08±0.02 1.25±0.04 阴性对照组 130 0.96±0.01** 1.15±0.02** 实验组 130 0.27±0.05**△△ 0.31±0.03**△△ F — 24 843.00 35 844.14 P — < 0.01 < 0.01 MS组内 — 0.001 0.001 q检验:与空白对照组比较** P < 0.01;与阴性对照组比较△△ P < 0.01 表 4 STAT3-shRNA对MDA-MB-231增殖率的影响(x ± s)
分组 n 细胞增殖活性 空白对照组 130 1.15±0.02 阴性对照组 130 1.23±0.03** 实验组 130 0.36±0.01**△△ F — 64 415.00 P — < 0.01 MS组内 — 0.001 q检验:与空白对照组比较** P < 0.01;与阴性对照组比较△△ P < 0.01 表 5 STAT3对MDA-MB-231侵袭和迁移能力的影响(x ± s)
分组 n 穿膜细胞数量(侵袭) 穿膜细胞数量(迁移) 空白对照组 130 186.0±5.4 115.6±4.3 阴性对照组 130 158.8±6.5** 117.9±3.6** 实验组 130 36.4±6.2**△△ 32.5±2.3**△△ F — 22 545.42 25 129.67 P — < 0.01 < 0.01 MS组内 — 36.617 12.246 q检验:与空白对照组比较** P < 0.01;与阴性对照组比较△△ P < 0.01 -
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