H-Ferritin-Regulated MicroRNAs Modulate Gene Expression in K562 Cells

In a previous study, we showed that the silencing of the heavy subunit (FHC) offerritin, the central iron storage molecule in the cell, is accompanied by a modification in global gene expression. In this work, we explored whether different FHC amounts might modulate miRNA expression levels in K562 cells and studied the impact of miRNAs in gene expression profile modifications. To this aim, we performed a miRNA-mRNA integrative analysis in K562 silenced for FHC (K562shFHC) comparing it with K562 transduced with scrambled RNA (K562shRNA). Four miRNAs, namely hsa-let-7g, hsa-let-7f, hsa-let-7i and hsa-miR-125b, were significantly up-regulated in silenced cells. The remarkable down-regulation of these miRNAs, following FHC expression rescue, supports a specific relation between FHC silencing and miRNA-modulation. The integration of target predictions with miRNA and gene expression profiles led to the identification of a regulatory network which includes the miRNAs up-regulated by FHC silencing, as well as91 down-regulated putative target genes. These genes were further classified in 9 networks; the highest scoring network, “Cell Death and Survival, Hematological System Development and Function, Hematopoiesis”, is composed by 18 focus molecules including RAF1 and ERK1/2. We confirmed that, following FHC silencing, ERK1/2 phosphorylation is severely impaired and that RAF1 mRNA is significantly down-regulated. Taken all together, our data indicate that, in our experimental model, FHC silencing may affect RAF1/pERK1/2 levels through the modulation of a specific set of miRNAs and add new insights in to the relationship among iron homeostasis and miRNAs.

既往研究表明，敲低细胞内核心铁存储分子铁蛋白重链（ferritin heavy chain, FHC）会伴随全基因组基因表达的改变。本研究旨在探究不同FHC表达水平是否可调控K562细胞中的微小RNA（microRNA, miRNA）表达水平，并分析miRNA对基因表达谱改变的影响。为此，我们对FHC敲低的K562细胞（K562shFHC）与转染乱序RNA的K562细胞（K562shRNA）开展了miRNA与mRNA整合分析。结果显示，敲低细胞中有4种miRNA显著上调，分别为hsa-let-7g、hsa-let-7f、hsa-let-7i与hsa-miR-125b。当恢复FHC表达后，上述miRNA的表达水平显著下调，这一结果证实FHC敲低与miRNA调控之间存在特异性关联。通过将靶基因预测结果与miRNA及基因表达谱进行整合分析，我们构建了包含FHC敲低上调的miRNA及其91个潜在下调靶基因的调控网络。我们将这91个靶基因进一步划分为9个功能网络，其中得分最高的网络为“细胞死亡与存活、造血系统发育与功能、造血作用”，该网络包含18个核心分子，包括RAF1与ERK1/2。我们进一步验证发现，FHC敲低后ERK1/2的磷酸化水平显著受损，且RAF1的mRNA表达量显著下调。综上，本实验数据表明，在本研究的细胞模型中，FHC敲低可通过调控特定miRNA群体影响RAF1/pERK1/2的表达水平，为铁稳态与miRNA之间的关联提供了新的研究视角。