Protein lysate microarray analysis to identify microRNAs regulating estrogen receptor signaling in breast cancer cell lines
Protein lysate microarray analysis to identify microRNAs regulating estrogen receptor signaling in breast cancer cell lines
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DOI：
10.1038/onc.2009.241
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发表时间：
2009-11-01
期刊：
影响因子：
8
通讯作者：
Kallioniemi, O.
中科院分区：
文献类型：
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作者：
Leivonen, S-K;Makela, R.;Kallioniemi, O.
由于microRNA在转录和翻译水平上的调节作用不同，预测其对靶蛋白的影响具有挑战性。在这项研究中，我们应用了一种新的蛋白质裂解物微阵列（LMA）技术，系统地监测319个pre-miR高通量转染到乳腺癌细胞后的靶蛋白水平。我们鉴定了21种下调雌激素受体α（ER α）的miRNAs，通过Western印迹和定量真实的时间PCR验证，并通过证明雌激素刺激的细胞生长的抑制。5种有效的ER α调节miRNA，miR-18 a、miR-18 b、miR-193 b、miR-206和miR-302 c，在3 '-非翻译区报告基因测定中被证实直接靶向ER α。他们抑制的基因表达特征与针对ER α的小干扰RNA的基因表达特征高度重叠，并且在所有测试的特征中，与已知雌激素诱导基因的抑制最密切相关。此外，与ER α阳性临床肿瘤相比，miR-18 a和miR-18 b在ER α阴性临床肿瘤中显示出更高的表达水平。总之，我们提供了miRNAs抑制乳腺癌ER α信号传导的系统和直接的功能证据，并证明了高通量LMA技术是一种新的，强大的技术，可以确定各种miRNAs对关键靶蛋白和相关细胞过程和途径的相对影响。Oncogene（2009）28，3926-3936; doi：10.1038/onc.2009.241; 2009年8月17日在线发表
Predicting the impact of microRNAs (miRNAs) on target proteins is challenging because of their different regulatory effects at the transcriptional and translational levels. In this study, we applied a novel protein lysate microarray (LMA) technology to systematically monitor for target protein levels after high-throughput transfections of 319 pre-miRs into breast cancer cells. We identified 21 miRNAs that downregulated the estrogen receptor-alpha (ER alpha), as validated by western blotting and quantitative real time-PCR, and by demonstrating the inhibition of estrogen-stimulated cell growth. Five potent ER alpha-regulating miRNAs, miR-18a, miR-18b, miR-193b, miR-206 and miR-302c, were confirmed to directly target ER alpha in 3'-untranslated region reporter assays. The gene expression signature that they repressed highly overlapped with that of a small interfering RNA against ER alpha, and across all the signatures tested, was most closely associated with the repression of known estrogen-induced genes. Furthermore, miR-18a and miR-18b showed higher levels of expression in ER alpha-negative as compared with ER alpha-positive clinical tumors. In summary, we present systematic and direct functional evidence of miRNAs inhibiting ER alpha signaling in breast cancer, and demonstrate the high-throughput LMA technology as a novel, powerful technique in determining the relative impact of various miRNAs on key target proteins and associated cellular processes and pathways. Oncogene (2009) 28, 3926-3936; doi:10.1038/onc.2009.241; published online 17 August 2009