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microRNA-374b抑制乳腺恶性肿瘤细胞增殖和转移的作用机制 被引量：1

MicroRNA-374b modulate cell growth and tumorigenesis of breast carcinoma through down-reg ulating monocy techemoattractant protein-1 exp ression
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摘要 目的探讨microRNA-374b抑制乳腺恶性肿瘤细胞增殖和转移的作用机制。方法构建microRNA-374b过表达的稳转MDA-MB-231细胞和空载MDA-MB-231细胞。通过MTS细胞增殖实验、单克隆形成实验、细胞划痕实验和细胞凋亡检测,计算细胞增殖相对数、划痕修复百分比和细胞凋亡百分比。通过microRNAs专业网站的预测筛选和双荧光素实验分析其可能的下游基因。采用实时定量PCR检测乳腺癌组织和癌周组织中人单核粒细胞趋化因子(MCP)-1和microRNA-374b含量。结果乳腺癌组织中的microRNA-374b相对表达量为0.43±0.03,显著低于癌周组织中的1.15±0.04(P<0.05)。MDA-MB-231-2396细胞的microRNA-374b相对表达量为0.53±0.20,显著高于MDA-MB-231-NC细胞的0.18±0.02(P<0.05)。培养第5、7天时MDA-MB-231-2396细胞增殖相对数分别为9.00±0.74、16.06±0.23,显著低于MDAMB-231-NC细胞的14.23±0.55、31.92±2.56(P值均<0.05)。MDA-MB-231-2396细胞结晶紫洗脱液的光密度值为0.54±0.02,显著低于MDA-MB-231-NC细胞的1.35±0.05(P<0.05)。MDA-MB-231-2396细胞早期凋亡百分比为(44.92±1.56)%,显著高于MDA-MB-232-NC细胞的(2.60±0.04)%(P<0.05)。MDA-MB-231-2396细胞于培养16、24、48h时的划痕修复百分比分别为(13.27±5.72)%、(18.25±8.43)%、(28.22±9.46)%,均显著低于MDA-MB-232-NC细胞的(25.48±0.53)%、(47.35%±1.40)%、(80.23±2.61)%(P值均<0.05)。双荧光素实验结果显示,MCP-1 3’UTR野生型与microRNA-374b类似物组合转染入293T细胞后两种荧光素的比例显著低于MCP-1 3’UTR突变型与microRNA-374b类似物组合(P<0.05),MCP-1 3’UTR野生型与microRNA-374b抑制物组合转染入293T细胞后两种荧光素的比例显著高于MCP-1 3’UTR突变型分别与microRNA抑制物的对照物、microRNA-374b、microRNA-374b类似物的对照物组合(P值均<0.05)。乳腺癌组织中MCP-1的相对表达量为5.79±1.32,显著高于癌周组织的0.07±0.01(P<0.05)。结论microRNA-374b可通过抑制MCP-1的表达来抑制乳腺恶性肿瘤细胞的增殖和转移能力,并促进其凋亡。 Objective To explore the role of microRNA-374b in the suppression of cell growth and metastasis of breast carcinoma. Methods MDA-MB-231 cells with over-expression of microRNA-374b and no- load control MDA-MB-231 cells were constructed. MTS cell proliferation assay, spheroid forming assay, wound- healing assay and cell apoptosis analysis were used to calculate the relative number of cell proliferation, percentage of wound repair and cell apoptosis in breast malignant cancer. Download target genes were predicted by three different microRNA specific websites and verified by double luciferin assay. The contents of monocyte chemoattractant protein-1 （MOP-1） and microRNA-374b were detected by real-time polymerase chain reaction（PCR）. Results The expression of microRNA-374b in breast cancer tissues was significantly lower than that in surrounding of breast cancer tissues （[0.43 ±. 0. 031 vs. [ 1.15 ±. 0. 041, P〈0.05）. The expression of microRNA- 374b in MDA-MB-231-2396 cells was significantly higher than that in MDA-MB-231-NC cells （[0.53 ± 0. 201 vs. [0.18＋0. 021, P〈0.05）. The relative abundance of proliferation in MDA-MB-231-NC cells was significantly higher than that in MDA-MB-231-2396 cells （[14.23±0.55] vs. [9.00±0.74], [31.92±2.56] vs. [16.06＋ 0.231, P〈0.05） in 5 and 7 day after vaccination. Optical density value of crystal violet eluent of spheroid in MDA-MB-231-2396 cells were significantly lower than that in MDA-MB-231-NC cell （E0.54 ± 0. 02] vs. E. 35 ± 0. 051, P〈0.05）, while early apoptosis percentage of MDA-MB-231-2396 cells was significantly higher than that of MDA-MB-231-NO cells （[44.92 4± 1.56] % vs. [2.60 ± 0.04-] %, P〈0.05）. The percentage of scratch repair in MDA-MB-231-2396 cells was significantly lower than that in MDA-MB-231-NC cells in 16 h, 24 h and 48 h after vaccination （[13.27±5.721% vs. [25.48±0.53]%,[18. 25± 8. 43] % vs. [47.35%±1.40]%, [28.22±9.461% vs. E80.23±2.61]%, all P〈0.05）. The results of double luciferin assay showed that the ratio of two luciferins in 293T cells with con-transfected MCP-1 3＇ UTR wt and microRNA-374b mimic was significantly lower than that of con-transfected MCP-1 3＇ UTR wt and microRNA-374b control （P〈0. 05）, while the ratio of two luciferins in 293T cells with the ratio of two luciferins in 293T cells with con-transfected MOP-1 3＇ UTR wt and anti microRNA-374b was significantly higher than that of con-transfected MOP-1 3＇ UTR wt with anti microRNA control or microRNA-374b or microRNA-374b control （all P〈0. 05）. The expression of MCP-1 in breast cancer tissues was significantly higher than that in surrounding of breast cancer tissues （[5.79±1. 321 vs. E[5. 07 ± 0. 01], P〈 0.05）. Conclusion MicroRNA-374b can suppress the proliferation and metastasis of breast carcinoma cells, which is accomplished by repressing MOP-1 expression. （Shanghai Med J, 2015, 38= 220-225）
作者 吕明丽 牛建梅 曾敏 余飞
机构地区 上海交通大学医学院附属国际和平妇幼保健院超声科 同济大学附属第十人民医院核医学科
出处 《上海医学》 CAS CSCD 北大核心 2015年第3期220-225,I0002,共7页 Shanghai Medical Journal
基金 中国福利会国际和平妇幼保健院优秀青年培育计划 国家自然科学基金(81301993)资助项目
关键词 microRNA-374b 人单核细胞趋化蛋白-1 乳腺肿瘤细胞 增殖 转移 MicroRNA-374b Monocyte chemoattractant protein-I Breast carcinoma Proliferation Metastasis
分类号 R737.9 [医药卫生—肿瘤]
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参考文献16

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二级参考文献19

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上海医学
2015年 第3期

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