Division of Molecular Genome Analysis, German Cancer Research Center, Heidelberg, Germany.
Oncogene. 2010 Jul 29;29(30):4297-306. doi: 10.1038/onc.2010.201. Epub 2010 May 31.
The genes encoding microRNAs of the human miR-200 family map to fragile chromosomal regions and are frequently downregulated upon tumor progression. Although having been reported to regulate epithelial-to-mesenchymal transition and transforming growth factor-beta-driven cell invasion, the role of the miR-200 family in EGF-driven breast cancer cell invasion, viability, apoptosis and cell cycle progression is still unknown. In particular, there is no study comparing the roles of the two clusters of this miRNA family. In this study, we show for the first time that miR-200 family members differentially regulate EGF-driven invasion, viability, apoptosis and cell cycle progression of breast cancer cells. We showed that, all miR-200 family members regulate EGF-driven invasion, with the miR-200bc/429 cluster showing stronger effects than the miR-200a/141 cluster. Furthermore, expression of the miR-200a/141 cluster results in G1 arrest supported by increased p27/Kip1 and decreased cyclin dependent kinase 6 expression. In contrast, expression of the 200bc/429 cluster decreases G1 population and increases G2/M phase, in line with the observed reduction of p27/Kip1 and upregulation of the inhibitory phosphorylation of Cdc25C, respectively. To test the hypothesis that phenotypical differences observed between the two clusters are caused by differential targeting spectrums, we performed genome-wide microarray profiling in combination with gain-of-function studies. This identified phospholipase C gamma 1 (PLCG1), which was downregulated only by the miR-200bc/429 cluster, as a potential candidate contributing to these phenotypical differences. Luciferase reporter assays validated PLCG1 as a direct functional target of miR-200bc/429 cluster, but not of miR-200a/141 cluster. Finally, loss of PLCG1 in part mimicked the effect of miR-200bc/429 overexpression in viability, apoptosis and EGF-driven cell invasion of breast cancer cells. Our results suggest that the miR-200 family has a tumor-suppressor function by negatively regulating EGF-driven cell invasion, viability and cell cycle progression in breast cancer.
miR-200 家族的人类 microRNA 基因定位于脆性染色体区域，并且在肿瘤进展过程中经常下调。尽管已经报道它们可以调节上皮-间充质转化和转化生长因子-β驱动的细胞侵袭，但 miR-200 家族在 EGF 驱动的乳腺癌细胞侵袭、活力、凋亡和细胞周期进展中的作用仍然未知。特别是，目前还没有研究比较该 miRNA 家族两个簇的作用。在这项研究中，我们首次表明 miR-200 家族成员可差异调节 EGF 驱动的乳腺癌细胞侵袭、活力、凋亡和细胞周期进展。我们表明，miR-200 家族成员均调节 EGF 驱动的侵袭，其中 miR-200bc/429 簇的作用强于 miR-200a/141 簇。此外，miR-200a/141 簇的表达导致通过增加 p27/Kip1 和降低细胞周期蛋白依赖性激酶 6 表达而导致 G1 期停滞。相比之下，表达 200bc/429 簇会减少 G1 群体并增加 G2/M 期，与观察到的 p27/Kip1 减少和 Cdc25C 的抑制性磷酸化上调一致。为了测试两个簇之间观察到的表型差异是由不同的靶向谱引起的假设，我们进行了全基因组微阵列分析并结合了功能获得研究。这确定了仅由 miR-200bc/429 簇下调的磷脂酶 Cγ1（PLCG1）作为导致这些表型差异的潜在候选物。荧光素酶报告基因测定验证了 PLCG1 是 miR-200bc/429 簇的直接功能靶标，但不是 miR-200a/141 簇的靶标。最后，PLCG1 的缺失部分模拟了 miR-200bc/429 过表达在乳腺癌细胞活力、凋亡和 EGF 驱动的细胞侵袭中的作用。我们的结果表明，miR-200 家族通过负向调节 EGF 驱动的乳腺癌细胞侵袭、活力和细胞周期进展，具有肿瘤抑制功能。
