Department of Molecular Biology, Princeton University, Princeton, New Jersey, United States of America.
PLoS One. 2012;7(2):e31240. doi: 10.1371/journal.pone.0031240. Epub 2012 Feb 13.
Changes in microRNA expression have been linked to a wide array of pathological states. However, little is known about the regulation of microRNA expression. The let-7 microRNA is a tumor suppressor that inhibits cellular proliferation and promotes differentiation, and is frequently lost in tumors. We investigated the transcriptional regulation of two let-7 family members, let-7a-3 and let-7b, which form a microRNA cluster and are located 864 bp apart on chromosome 22q13.31. Previous reports present conflicting data on the role of the NF-κB transcription factor in regulating let-7. We cloned three fragments upstream of the let-7a-3/let-7b miRNA genomic region into a plasmid containing a luciferase reporter gene. Ectopic expression of subunits of NF-κB (p50 or p65/RelA) significantly increased luciferase activity in HeLa, 293, 293T and 3T3 cells, indicating that the let-7a-3/let-7b promoter is highly responsive to NF-κB. Mutation of a putative NF-κB binding site at bp -833 reduced basal promoter activity and decreased promoter activity in the presence of p50 or p65 overexpression. Mutation of a second putative binding site, at bp -947 also decreased promoter activity basally and in response to p65 induction, indicating that both sites contribute to NF-κB responsiveness. While the levels of the endogenous primary let-7a and let-7b transcript were induced in response to NF-κB overexpression in 293T cells, the levels of fully processed, mature let-7a and let-7b miRNAs did not increase. Instead, levels of Lin-28B, a protein that blocks let-7 maturation, were induced by NF-κB. Increased Lin-28B levels could contribute to the lack of an increase in mature let-7a and let-7b. Our results suggest that the final biological outcome of NF-κB activation on let-7 expression may vary depending upon the cellular context. We discuss our results in the context of NF-κB activity in repressing self-renewal and promoting differentiation.
miRNA 表达的变化与广泛的病理状态有关。然而，miRNA 表达的调控机制知之甚少。let-7 miRNA 是一种肿瘤抑制因子，可抑制细胞增殖并促进分化，且在肿瘤中经常丢失。我们研究了两个 let-7 家族成员（let-7a-3 和 let-7b）的转录调控，这两个成员形成一个 miRNA 簇，位于染色体 22q13.31 上相距 864bp 的位置。先前的报告对 NF-κB 转录因子在调节 let-7 中的作用提出了相互矛盾的数据。我们将 let-7a-3/let-7b miRNA 基因组区域上游的三个片段克隆到含有荧光素酶报告基因的质粒中。NF-κB 亚基（p50 或 p65/RelA）的异位表达显著增加了 HeLa、293、293T 和 3T3 细胞中的荧光素酶活性，表明 let-7a-3/let-7b 启动子对 NF-κB 高度敏感。在 bp-833 处的一个假定的 NF-κB 结合位点发生突变后，基础启动子活性降低，并且在 p50 或 p65 过表达时，启动子活性降低。在 bp-947 处的第二个假定结合位点发生突变也会降低基础和 p65 诱导的启动子活性，表明这两个位点均有助于 NF-κB 反应性。虽然内源性初级 let-7a 和 let-7b 转录物的水平在 293T 细胞中 NF-κB 过表达时被诱导，但完全加工的成熟 let-7a 和 let-7b miRNA 的水平并未增加。相反，Lin-28B 的水平升高，Lin-28B 是一种阻止 let-7 成熟的蛋白质。NF-κB 诱导的 Lin-28B 水平升高可能导致成熟 let-7a 和 let-7b 的水平没有增加。我们的结果表明，NF-κB 激活对 let-7 表达的最终生物学结果可能因细胞环境而异。我们根据 NF-κB 活性在抑制自我更新和促进分化方面的作用讨论了我们的结果。
