Sachdeva Mohit, Dodd Rebecca D, Huang Zhiqing, Grenier Carole, Ma Yan, Lev Dina C, Cardona Diana M, Murphy Susan K, Kirsch David G
Department of Radiation Oncology, Duke University Medical Center, Durham, NC 27710, USA.
Department of Obstetrics and Gynecology, Duke University Medical Center, Durham, NC 27710, USA.
Cancer Lett. 2015 Dec 1;369(1):202-11. doi: 10.1016/j.canlet.2015.08.016. Epub 2015 Aug 24.
Accumulating evidence indicates that microRNAs (miRs) regulate cancer metastasis. We have shown that miR-182 drives sarcoma metastasis in vivo by coordinated regulation of multiple genes. Recently, we also demonstrated that in a subset of primary sarcomas that metastasize to the lung, miR-182 expression is elevated through binding of MyoD1 to the miR-182 promoter. However, it is not known if there are also transcription factors that inhibit miR-182 expression. Defining negative regulators of miR-182 expression may help explain why some sarcomas do not metastasize and may also identify pathways that can modulate miR-182 for therapeutic benefit. Here, we use an in silico screen, chromatin-immunoprecipitation, and luciferase reporter assays to discover that Kruppel like factor-3 (Klf-3) is a novel transcriptional repressor of miR-182. Knockdown of Klf-3 increases miR-182 expression, and stable overexpression of Klf-3, but not a DNA-binding mutant Klf-3, decreases miR-182 levels. Klf-3 expression is downregulated in both primary mouse and human metastatic sarcomas, and Klf-3 levels negatively correlate with miR-182 expression. Interestingly, Klf-3 also negatively regulates MyoD1, suggesting an alternative mechanism for Klf-3 to repress miR-182 expression in addition to direct binding of the miR-182 promoter. Using Methylation Specific PCR (MSP) and pyrosequencing assays, we found that Klf-3 is epigenetically silenced by DNA hypermethylation both in mouse and human sarcoma cells. Finally, we show the DNA methylation inhibitor 5'Azacytidine (Aza) restores Klf-3 expression while reducing miR-182 levels. Thus, our findings suggest that demethylating agents could potentially be used to modulate miR-182 levels as a therapeutic strategy.
越来越多的证据表明，微小RNA（miR）可调节癌症转移。我们已经证明，miR-182通过对多个基因的协同调控在体内驱动肉瘤转移。最近，我们还证明，在转移至肺部的原发性肉瘤亚组中，MyoD1与miR-182启动子结合可使miR-182表达升高。然而，尚不清楚是否也存在抑制miR-182表达的转录因子。确定miR-182表达的负调节因子可能有助于解释为何有些肉瘤不会发生转移，还可能识别出可调节miR-182以获得治疗益处的途径。在此，我们通过计算机筛选、染色质免疫沉淀和荧光素酶报告基因检测发现，Kruppel样因子3（Klf-3）是miR-182的新型转录抑制因子。敲低Klf-3可增加miR-182表达，而稳定过表达Klf-3（而非DNA结合突变体Klf-3）可降低miR-182水平。在原发性小鼠和人类转移性肉瘤中，Klf-3表达均下调，且Klf-3水平与miR-182表达呈负相关。有趣的是，Klf-3也对MyoD1起负调节作用，这表明除了直接结合miR-182启动子外，Klf-3还存在另一种抑制miR-182表达的机制。使用甲基化特异性PCR（MSP）和焦磷酸测序检测，我们发现Klf-3在小鼠和人类肉瘤细胞中均因DNA高甲基化而发生表观遗传沉默。最后，我们发现DNA甲基化抑制剂5'-氮杂胞苷（Aza）可恢复Klf-3表达，同时降低miR-182水平。因此，我们的研究结果表明，去甲基化剂有可能作为一种治疗策略用于调节miR-182水平。
