| 张振华,阚云珍,苏自杰.miR-139通过下调CXCR4/CXCL12生物轴抑制乳腺癌定向转移的分子机制研究[J].中国肿瘤,2018,27(1):61-67. |
| miR-139通过下调CXCR4/CXCL12生物轴抑制乳腺癌定向转移的分子机制研究 |
| Molecular Mechanism of MiR-139 Inhibiting Directional Metastasis of Breast Cancer by Down-regulating CXCR4-CXCL12 Bio-axis |
| 投稿时间：2017-04-07 |
| DOI：10.11735/j.issn.1004-0242.2018.01.A009 |
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| 中文关键词: 乳腺癌 miRNA-139 CXCR4/CXCL12 定向转移 |
| 英文关键词:beast cancer miRNA-139 CXCR4/CXCL12 directed metastasis |
| 基金项目:2013年河南省重点科技攻关计划项目（132102310095） |
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| 中文摘要: |
| 摘 要：［目的］ 检测miR-139在乳腺癌细胞系及组织中的表达，验证CXCR4是否为miR-139直接调控的靶基因，并探究miR-139是否可通过靶向抑制CXCR4/CXCL12生物轴从而抑制乳腺癌细胞的定向转移。［方法］ 实时荧光定量聚合酶链式反应（qRT-PCR）用于检测miR-139在34对转移性乳腺癌患者的原发灶、转移灶及癌旁组织中，在5株乳腺癌细胞系及1株正常乳腺上皮细胞中的表达。将包含miR-139的重组慢病毒质粒及其阴性对照空载体质粒vector分别以病毒/细胞数量=20的比例感染MDA-MB-231细胞，经2.0μg/ml嘌呤霉素筛选，成功构建稳定表达miR-139或vector的MDA-MB-231细胞，将MDA-MB-231vector和MDA-MB-231miR-139细胞分别经尾静脉注射到BALB/c裸鼠中，构建乳腺癌裸鼠转移模型，观察过表达miR-139对裸鼠体内转移能力的影响。TargetScan软件用于预测miR-139的靶基因，根据预测结果，将野生型或突变型CXCR4的3’-UTR区域克隆到荧光素酶报告基因的下游（CXCR4-wt或CXCR4-mut）并分别与miR-139 mimics或scramble共转染后检测荧光素酶的活性。qRT-PCR和Western blot分别检测转染miR-139 mimics和scramble后CXCR4、CXCL12的mRNA和蛋白表达。［结果］ miR-139在转移性乳腺癌原发灶和转移灶中的表达均低于癌旁组织，且转移灶中表达最低，miR-139在乳腺癌细胞系中的表达水平显著低于正常乳腺上皮细胞MCF-10A，且MDA-MB-231细胞中最低。尾静脉注射MDA-MB-231miR-139的裸鼠肺转移灶的个数均显著低于MDA-MB-231vector细胞。TargetScan软件预测CXCR4为miR-139直接调控的靶基因，CXCR4-wt+miR-139共转染组比CXCR4-wt+scramble共转染组的荧光素酶活性强度显著降低，CXCR4-mut+miR-139共转染组和CXCR4-wt+scramble共转染组间荧光素酶活性强度无显著性差异。转染miR-139后，CXCR4和CXCL12的mRNA和蛋白表达水平均显著下调，CXCR4/CXCL12生物轴下游的p-AKT和p-ERK蛋白表达水平也显著下调。［结论］ miR-139可通过靶向调控CXCR4/CXCL12生物轴而抑制乳腺癌细胞MDA-MB-231定向转移。 |
| 英文摘要: |
| Abstract：［Purpose］To investigate the effect of miR-139 on directional metastasis of breast cancer (BC) and related molecular mechanism. ［Methods］ qRT-PCR was used to detect the expression of miR-139 in the primary，metastatic cancer and adjacent tissues of 34 patients with metastatic breast cancer and 5 breast cancer cell lines and 1 normal mammary epithelial cell line MCF-10A. Breast cancer MDA-MB-231 cells were infected with recombinant lentivirus plasmid containing miR-139 and its negative control vector to construct stable MDA-MB-231miR-139 and MDA-MB-231vector cells. The two stable cells were injected into BALB/c nude mice via tail vein to construct metastatic model of breast cancer，respectively. And the effect of miR-139 expression on metastasis in nude mice was observed. TargetScan was used to predict the target gene of miR-139，and the 3′-UTR region of wild-type or mutant CXCR4 was cloned into downstream of the luciferase reporter gene(CXCR4-wt or CXCR4-mut) and tested for luciferase activity after co-transfection into MDA-MB-231 cells with miR-139 mimics or scramble. MDA-MB-231 cells were transfected with miR-139 mimics and scramble，the mRNA and protein expressions of CXCR4 and CXCL12 were detected by qRT-PCR and Western blot methods. ［Results］ The expression of miR-139 was lower in primary and metastatic lesions than that in adjacent tissues，and lowest in metastatic lesions.The expression of miR-139 in breast cancer cell lines was significantly lower than that in normal mammary epithelial MCF-10A cells，and lowest in MDA-MB-231 cells. The number of metastatic foci formed by MDA-MB-231miR-139 in nude mice were significantly lower than that of MDA-MB-231vector cells. TargetScan predicted that CXCR4 was the direct target of miR-139. miR-139 significantly suppressed the luciferase activity in CXCR4-wt+miR-139 co-transfection group than CXCR4-wt+scramble group. There was no significant difference in luciferase activity between CXCR4-mut+miR-139 and CXCR4-wt+scramble co-transfection group. The mRNA and protein expression levels of CXCR4 and CXCL12 were significantly reduced in MDA-MB-231 cells after transfection of miR-139，the expression levels of p-AKT and p-ERK proteins，which were the downstream of CXCR4/CXCL12 bio-axis，were also significantly down-regulated after transfection of miR-139. ［Conclusion］ MiR-139 can inhibit the directional metastasis of breast cancer MDA-MB-231 cells by targeting CXCR4/CXCL12 bio-axis. |
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