Department of Biochemical Engineering and Biotechnology; Indian Institute of Technology; Delhi, India.
RNA Biol. 2013 Aug;10(8):1283-90. doi: 10.4161/rna.25482. Epub 2013 Jun 20.
MicroRNAs and AU Rich element (ARE)-mediated degradation of transcripts are thought to be two independent means of gene regulation at the post-transcriptional level. However, since their site of action is the same (3'UTR of mRNA), there exists a high probability that specific miRNAs may bind to AREs and, thus, interact with ARE-binding proteins (ARE-BPs) to regulate transcript levels. In this study, we have characterized AREs as potential targets of hsa-miR-3134. An analysis of the global gene expression profile of breast cancer cell line MCF7 overexpressing miR-3134 revealed the presence of at least one AUUUA element in the 3'-UTRs of 63% of miR-3134 regulated protein coding genes. Quantitative RT-PCR or 3'UTR luciferase assays show that miR-3134 mediates an up to 4-8-fold increase in the levels of ARE bearing transcripts-SOX9, VEGFA, and EGFR, while mutated miR-3134 shows a decreased effect. The miR-3134-mediated increase in transcript levels was unaffected by treatment with transcription inhibitor (actinomycin D), indicating that miR-3134 enhances transcript stability. To investigate a possible interplay between miR-3134 and a prototype ARE-BP, HuR, we compared their overexpression transcriptome profiles. Interestingly, up to 80% of miR-3134-regulated genes were also regulated by HuR. Overexpression studies of HuR alone or in combination with miR-3134 shows that wt miR-3134 but not a mutated miR-3134 promotes stabilization of HuR-regulated transcripts SOX9, VEGFA, and EGFR as confirmed by qRT-PCR or RNA-immunoprecipitation experiments. Overall, this report suggests that collaboration between ARE-binding microRNAs and ARE-binding proteins could be a general mechanism of 3'-UTR mediated regulation of gene expression in human cells.
MicroRNAs 和 AU 丰富元素 (ARE)-介导的转录物降解被认为是转录后水平基因调控的两种独立方式。然而，由于它们的作用位点相同（mRNA 的 3'UTR），特定的 microRNAs 很有可能与 ARE 结合，并与 ARE 结合蛋白 (ARE-BP) 相互作用，从而调节转录物水平。在这项研究中，我们将 ARE 鉴定为 hsa-miR-3134 的潜在靶标。对过表达 miR-3134 的 MCF7 乳腺癌细胞系的全基因表达谱进行分析，结果表明 miR-3134 调控的蛋白编码基因中有至少 63%的 3'UTR 中存在一个 AUUUA 元件。定量 RT-PCR 或 3'UTR 荧光素酶测定显示，miR-3134 介导 ARE 携带的转录物-SOX9、VEGFA 和 EGFR 的水平增加了 4-8 倍，而突变 miR-3134 的作用降低。miR-3134 介导的转录物水平增加不受转录抑制剂（放线菌素 D）处理的影响，表明 miR-3134 增强了转录物的稳定性。为了研究 miR-3134 与原型 ARE-BP HuR 之间可能存在的相互作用，我们比较了它们的过表达转录组谱。有趣的是，高达 80%的 miR-3134 调控基因也受到 HuR 的调控。单独过表达 HuR 或与 miR-3134 联合过表达研究表明，wt miR-3134 而非突变 miR-3134 可促进 HuR 调控的转录物 SOX9、VEGFA 和 EGFR 的稳定，这通过 qRT-PCR 或 RNA-免疫沉淀实验得到证实。总的来说，本报告表明，ARE 结合 microRNAs 和 ARE 结合蛋白之间的协作可能是人类细胞 3'UTR 介导的基因表达调控的一般机制。
