鉴定和表征人 miR-1908 启动子中的 NF-κB 结合位点。
Identification and characterization of NF-kappaB binding sites in human miR-1908 promoter.
机构信息
Nanjing Maternal and Child Health Institute, Nanjing Maternal and Child Health Care Hospital Affiliated to Nanjing Medical University, Nanjing,; Department of Pediatrics, Affiliated Hospital of Nantong University, Nantong, China.
Nanjing Maternal and Child Health Institute, Nanjing Maternal and Child Health Care Hospital Affiliated to Nanjing Medical University, Nanjing.
出版信息
Biomed Pharmacother. 2015 Aug;74:158-63. doi: 10.1016/j.biopha.2015.08.018. Epub 2015 Aug 15.
BACKGROUND
Previous studies reported that miR-1908 was highly expressed in mature human adipocytes. Adipokines tumor necrosis factor α (TNF-α) that was known to activate NF-kappaB signaling could affect the expression of miR-1908 in adipocytes. However, little is known about the underlying mechanisms.
METHODS
In this study, we identified miR-1908 promoter using polymerase chain reaction (PCR) from human genomic DNA. Bioinformatic analysis was applied to predict the NF-kappaB binding sites in miR-1908 promoter. Real-time PCR, dual luciferase reporter assay, Mutagenesis analysis and electrophoretic mobility shift assay (EMSA) were performed to demonstrate the function of NF-kappaB binding sites in miR-1908 promoter.
RESULTS
1243bp miR-1908 promoter located in the intron of host gene fatty acid desaturase 1 (FADS1). Bioinformatic analysis revealed the presence of two putative NF-kappaB binding sites. TNF-α restricts miR-1908 expression in preadipocytes, and TNF-α decreases miR-1908 promoter activity in HEK293T cells. In addition, those two NF-kappaB transcription factor binding sites in miR-1908 promoter were functional.
CONCLUSION
Our findings demonstrated that miR-1908 has its own transcription unit, and revealed the transcriptional mechanisms of miR-1908 expression based on NF-kappaB signaling. This study offers a theoretical basis for understanding the transcriptional mechanism of miR-1908 expression and may provide a new strategy for obesity clinical therapy.
背景
先前的研究表明，miR-1908 在成熟的人类脂肪细胞中高度表达。已知脂肪细胞因子肿瘤坏死因子-α（TNF-α）可激活 NF-κB 信号通路，从而影响 miR-1908 在脂肪细胞中的表达。然而，其潜在机制知之甚少。
方法
在这项研究中，我们使用聚合酶链反应（PCR）从人类基因组 DNA 中鉴定出 miR-1908 启动子。生物信息学分析用于预测 miR-1908 启动子中 NF-κB 结合位点。实时 PCR、双荧光素酶报告基因检测、突变分析和电泳迁移率变动分析（EMSA）用于证明 NF-κB 结合位点在 miR-1908 启动子中的功能。
结果
miR-1908 启动子位于宿主基因脂肪酸去饱和酶 1（FADS1）的内含子中，大小为 1243bp。生物信息学分析显示存在两个潜在的 NF-κB 结合位点。TNF-α 限制前脂肪细胞中 miR-1908 的表达，并且 TNF-α 降低 HEK293T 细胞中 miR-1908 启动子活性。此外，miR-1908 启动子中的这两个 NF-κB 转录因子结合位点具有功能。
结论
我们的研究结果表明，miR-1908 具有其自身的转录单元，并揭示了基于 NF-κB 信号的 miR-1908 表达的转录机制。本研究为理解 miR-1908 表达的转录机制提供了理论依据，并可能为肥胖症的临床治疗提供新策略。
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