Laboratory of Integrated Biosciences, School of Life Sciences, Sun Yat-sen University, Guangzhou, China.
J Biol Chem. 2011 Nov 18;286(46):39703-14. doi: 10.1074/jbc.M111.293126. Epub 2011 Sep 8.
The human microRNA cluster MC-let-7a-1∼let-7d, with three members let-7a-1, let-7f-1, and let-7d, is an important cluster of the let-7 family. These microRNAs play critical roles in regulating development and carcinogenesis. Therefore, precise control of MC-let-7a-1∼let-7d level is critical for cellular functions. In this study, we first showed that the expression of these three members was significantly reduced in human hepatocellular carcinoma HepG2 cells as compared with the immortalized human liver L02 cells. We demonstrated that the MC-let-7a-1∼let-7d cluster was encoded by a single polycistronic transcript driven by a 10-kb upstream promoter, with two MYC-binding sites. Importantly, MYC inhibited MC-let-7a-1∼let-7d promoter activity via binding to the noncanonical E-box 3 downstream of the transcription start sites, whereas it enhanced promoter activity by binding to the canonical E-box 2 upstream of the transcription start sites. We found that although the binding affinity of MYC to E-box 2 was stronger than E-box 3, the binding quantum of MYC to E-box 3 was significantly higher in cancerous HepG2 cells as compared with the noncancerous L02 cells. In addition, forced expression of let-7 could reverse the MYC-mediated cell proliferation. These findings suggested that in L02 cells with a low level of MYC, MYC binds mainly to E-box 2 to enhance MC-let-7a-1∼let-7d expression. However, in HepG2 cells with an elevated MYC, the extra MYC could bind to E-box 3 to suppress the transcription of MC-let-7a-1∼let-7d and thus enable HepG2 cells to maintain a high level of MYC and a low level of let-7 microRNAs simultaneously.
人类 microRNA 簇 MC-let-7a-1∼let-7d，由三个成员 let-7a-1、let-7f-1 和 let-7d 组成，是 let-7 家族的一个重要簇。这些 microRNAs 在调节发育和致癌作用中发挥关键作用。因此，MC-let-7a-1∼let-7d 水平的精确控制对细胞功能至关重要。在这项研究中，我们首先表明，与永生化人肝 L02 细胞相比，这些三个成员在人肝癌 HepG2 细胞中的表达显著降低。我们证明，MC-let-7a-1∼let-7d 簇由一个单一的多顺反子转录物编码，由 10kb 上游启动子驱动，该启动子具有两个 MYC 结合位点。重要的是，MYC 通过结合转录起始位点下游的非典型 E-box3 抑制 MC-let-7a-1∼let-7d 启动子活性，而通过结合转录起始位点上游的典型 E-box2 增强启动子活性。我们发现，尽管 MYC 与 E-box2 的结合亲和力强于 E-box3，但在与非癌性 L02 细胞相比，癌性 HepG2 细胞中 MYC 与 E-box3 的结合量子显著更高。此外，let-7 的强制表达可以逆转 MYC 介导的细胞增殖。这些发现表明，在 MYC 水平较低的 L02 细胞中，MYC 主要结合 E-box2 以增强 MC-let-7a-1∼let-7d 的表达。然而，在 MYC 水平升高的 HepG2 细胞中，多余的 MYC 可以结合到 E-box3 上，从而抑制 MC-let-7a-1∼let-7d 的转录，从而使 HepG2 细胞能够同时维持高水平的 MYC 和低水平的 let-7 microRNAs。
