Human with-no-lysine kinase-4 3'-UTR acting as the enhancer and being targeted by miR-296.
Human with-no-lysine kinase-4 3'-UTR acting as the enhancer and being targeted by miR-296.
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DOI：
10.1016/j.biocel.2010.06.006
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发表时间：
2010-09
期刊：
影响因子：
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通讯作者：
J. Mao;Chunyi Li;Yuan-yuan Zhang;Yinghui Li;Yanyan Zhao
中科院分区：
文献类型：
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作者：
J. Mao;Chunyi Li;Yuan-yuan Zhang;Yinghui Li;Yanyan Zhao
人无赖氨酸激酶-4(HWNK4)是丝氨酸-苏氨酸蛋白激酶家族的一员，可能通过调节多种离子转运蛋白参与高血压的病理生理过程。我们感兴趣的是调控hWNK4表达的分子机制。3‘-非翻译区是基因调控的重要区域。在这里，我们集中在hWNK4的3‘-非编码区来研究它是如何调控hWNK4的表达的。荧光素酶分析表明，hWNK43 3‘-UTR显著增强了启动子的转录活性，无论是同源的还是异源的。染色体构象捕获分析进一步证明，3‘-UTR增强子通过与hWNK4启动子的远距离串扰，以细胞特异性的方式发挥作用。随着启动子的缺失，hWNK4 3‘-UTR的增强子作用减弱，表明启动子中的基序可能是hWNK4 3’-UTR功能所必需的。同时，hWNK43 3‘-UTR含有miR-296结合位点，通过生物信息学算法预测，并通过报告实验验证为真实靶点。实时定量聚合酶链式反应和Western印迹分析显示，miR-296可在转录后水平以细胞特异性模式下调hWNK4的表达。这些数据表明hWNK4 3‘-UTR通过与启动子的串扰发挥增强作用，而miR-296通过靶向其3’-UTR以细胞特异性的方式抑制hWNK4的表达。MiR-296和辅因子对3‘-UTR的协同调节可能与hWNK4的生理病理作用有关。
Human with-no-lysine kinase-4 (hWNK4) is a member of the serine–threonine protein kinase family and may be involved in pathophysiological processes of hypertension through regulating diverse ion transporters. We are interested in the molecular mechanism of regulating hWNK4 expression. The 3′-untranslated region (3′-UTR) is an important area for gene regulation. Here, we focused on the 3′-UTR of hWNK4 to investigate how it regulates hWNK4 expression. A luciferase assay showed that the hWNK4 3′-UTR obviously enhanced the transcriptional activity of the promoter, whether homologous or heterologous. Chromosome conformation capture assay further demonstrated that the 3′-UTR enhancer functioned through distant crosstalking with the hWNK4 promoter in a cell-specific manner. Following the deletion of the promoter, the enhancer action of the hWNK4 3′-UTR fell away, indicating that the motifs within the promoter could be essential for the function of the hWNK4 3′-UTR. The hWNK4 3′-UTR, meanwhile, contained a miR-296 binding site, predicted by bioinformatics algorithms and verified as a true target using a reporter assay. Expression of hWNK4 can be downregulated by miR-296 at the posttranscriptional level in cell-specific pattern, shown by real-time quantitative PCR and Western blot assays. These data demonstrate the hWNK4 3′-UTR plays an enhancer role by crosstalking with the promoter, and miR-296 suppresses hWNK4 expression through targeting on its 3′-UTR in a cell-specific fashion. The coordinated modulation of miR-296 and cofactors on the 3′-UTR may be attributed to hWNK4 physiopathological function.