低氧下miRNA对铁调节蛋白（IRPs）的调控研究

Iron is the essensial element in organism. Human body has a complex and rigorous iron regulatory system after a long time evolution. The concentration of oxygen plays an important role in the function of the body. Low oxygen may influence the expression of iron-related proteins involved in iron metabolism including IRPs. IRPs is also known as "the regulator of intracellular iron homeostasis".As a RNA-binding protein, IRPs regulate intracellular iron levels post-transcriptionally through binding with IREs of the target genes mRNA. MicroRNAs (miRNAs) are endogenous non-coding RNAs which are found in eukaryotes has rapidly emerged as one of the central negative regulators of expression of an extensive repertoire of genes. miRNA acts in a complex form of miRISC, then miRISC which binding of a miRNA move to its target mRNA through base-pairing mechanism with its 5' end 2 to 8 nts (termed as seed-site) complementary to recognition motif within the target mRNA. miRNAs can either inhibit translation(partial complementary) or induce degradation (full complementary) of its target mRNA, depending on the matches of the seed-site of miRNAs to its target. Recent studies have shown that miRNAs are not only involved in a variety of regulatory pathways of the body including growth and metabolism, but also play an important role under hypoxia. However, only a small portion of biological functions of miRNAs has been elucidated. The research about miRNAs on cellular iron homeostasis under hypoxia is rare. Our recent studies have shown that the cellular total iron increases under hypoxic conditions and the expression of IRPs is lower under long-time hypoxia than normoxia. Besides, we have identified 13 putative miRNAs of IRPs through bioinformatics prediction methods and biochip points. Therefore, this study attempts to prove the miRNA can participate in the post-transcription regulation of IRPs under hypoxia, find target miRNA of IRPs, study on the regulatory function of miRNA to IRPs, and further explore the impact of specific miRNA on intracellular iron homeostasis under hypoxia. The study could expand our knowledge about IRPs itself regulation mechanism and could provide a theoretical basis in genetic level of regulation of intracellular iron homeostasis. It is also possible to provide a new biological marker for the diagnosis of disorders of intracellular iron. Besides, it may play an important role or provide a new mean for the prevention and treatment of hypoxia/ischemia-induced iron disorders diseases in clinical cases.

铁是生命体的必需元素之一，人体拥有复杂而精密的铁调节系统。机体正常功能的发挥需要适当的氧气供给，低氧环境会导致铁调节系统中相关蛋白的变化并进一步影响铁稳态，其中包括胞内铁稳态调节者即铁调节蛋白（IRPs）。MicroRNAs（miRNAs）是在真核生物中发现的具有负调控功能的非编码RNA。miRNAs除参与细胞中的生长、代谢等基本调节途径外，在低氧应答中也扮演了重要角色。关于低氧下miRNAs对细胞铁稳态的影响报道较少。我们前期研究发现长时程低氧后IRPs蛋白显著降低，通过生物信息学预测及生物芯片初筛确定了13个低氧下IRPs的潜在特异性miRNA。本研究试图证明低氧下miRNA可参与IRPs的调控，通过确定IRPs特异性miRNA，探讨低氧下通过特定miRNA调控IRPs对胞内铁稳态的影响，为我们从基因水平调控胞内铁稳态的研究提供理论依据，该研究可能为胞内铁紊乱的诊断提供新的生物学标记。

本研究发现低氧能引起IRPs基因表达变化，虽然在不同类型的细胞上的结果不尽相同，但是大部分细胞上低氧后IRP1呈现出双向性变化，IRP2在低氧下表现相对稳定。低氧对IRP1的具体影响表现为在短时程低氧表现出IRP1mRNA表达下调，长时程低氧IRP1mRNA表达上升。在第一部分的研究中我们首先通过生物信息学分析潜在的低氧下可调控 IRPs 的特异性miRNA，通过芯片分析和实验筛选出has-miR-141-3p和has-miR-200a-3p对IRPs具有一定调节作用。通过双荧光素酶报告基因系统我们证实了has-miR-141-3p及has-miR-200a-3p可与IRPs上的MREs序列结合，并抑制IRPs的转录。但是miR-141-3p对IRP1的抑制作用更为明显。随后我们进一步通过体外转染实验证实在低氧3h后has-miR-141-3p可通过下调IRP1减少细胞铁摄入。第二部分的研究结果证明长时程低氧对IRPs基因的表达调控可能的机制，我们发现长时程低氧后pCREB的表达被激活，推测长时程IRP1mRNA的表达增强与pCREB的激活有关。通过western、PCR、EMSA和ChIP证实：长时程低氧可激活pCREB的表达，增加pCREB的核转位及与IRP1基因启动区的CREB反应元件结合，并诱导IRP1基因转录。综上，低氧对IRP的表达影响表现出双向性，短时程低氧可抑制IRP1mRNA的转录，其主要作用机制除了已知的低氧诱导低氧诱导因子表达后抑制IRP1基因表达外，还与低氧下has-miRNA-141-3p或has-miR-200a-3p表达上调抑制了IRP1 mRNA的转录有关。长时程低氧可促进IRP1mRNA的转录，其主要作用机制是长时程低氧激活了CREB的磷酸化促进其核转位，并结合到IRP1基因启动子区的CREB反应元件激活并诱导IRP1mRNA的转录。该研究有助于我们从分子水平了解低氧应激对细胞铁稳态的影响，为各类缺血/低氧性疾病以及铁相关的疾病的防治提供了一定的实验依据和策略。