shRNA targeting of ferritin heavy chain activates H19/miR-675 axis in K562 cells.

PURPOSE: The heavy subunit of the iron storage protein ferritin (FHC) is essential for the intracellular iron metabolism and, at the same time, it represents a central hub of iron-independent pathways, such as cell proliferation, angiogenesis, p53 regulation, chemokine signalling, stem cell expansion, miRNAs expression. In this work we have explored the ability of FHC to modulate gene expression in K562 cells, through the up-regulation of the lncRNA H19 and its cognate miR-675. MATERIALS AND METHODS: Targeted silencing of FHC was performed by lentiviral-driven shRNA strategy. FHC reconstitution was obtained by full length FHC cDNA transfection with Lipofectamine 2000. ROS amounts were determined with the redox-sensitive probe H2DCFDA. H19, miR-675, miR-107, Twist1, ID3, EPHB6, GNS, ANK1 and SMAD6 mRNA amounts were quantified by Taqman assay and qPCR analysis. RESULTS: FHC silencing in K562 cells modulates gene expression through the up-regulation of the lncRNA H19 and its cognate miR-675. Experimental findings demonstrate that the molecular mechanism underlying this phenomenon is represented by an FHC knock-down-triggered increase in reactive oxygen species (ROS) production. CONCLUSIONS: In this paper we uncover a so far not described function of the ferritin heavy subunit in the control of lncRNA pathways.

Epithelial-to-mesenchymal transition in FHC-silenced cells: the role of CXCR4/CXCL12 axis.

BACKGROUND: Ferritin plays a central role in the intracellular iron metabolism; the molecule is a nanocage of 24 subunits of the heavy and light types. The heavy subunit (FHC) is provided of a ferroxidase activity and thus performs the key transformation of iron in a non-toxic form. Recently, it has been shown that FHC is also involved in additional not iron-related critical pathways including, among the others, p53 regulation, modulation of oncomiRNAs expression and chemokine signalling. Epithelial to mesenchymal transition (EMT) is a cellular mechanism by which the cell acquires a fibroblast-like phenotype along with a decreased adhesion and augmented motility. In this work we have focused our attention on the role of the FHC on EMT induction in the human cell lines MCF-7 and H460 to elucidate the underlying molecular mechanisms. METHODS: Targeted silencing of the FHC was performed by lentiviral-driven shRNA strategy. Reconstitution of the FHC gene product was obtained by full length FHC cDNA transfection with Lipofectamine 2000. MTT and cell count assays were used to evaluate cell viability and proliferation; cell migration capability was assayed by the wound-healing assay and transwell strategy. Quantification of the CXCR4 surface expression was performed by flow cytometry. RESULTS: Experimental data indicated that FHC-silenced MCF-7 and H460 cells (MCF-7shFHC, H460shFHC) acquire a mesenchymal phenotype, accompanied by a significant enhancement of their migratory and proliferative capacity. This shift is coupled to an increase in ROS production and by an activation of the CXCR4/CXCL12 signalling pathway. We present experimental data indicating that the cytosolic increase in ROS levels is responsible for the enhanced proliferation of FHC-silenced cells, while the higher migration rate is attributable to a dysregulation of the CXCR4/CXCL12 axis. CONCLUSIONS: Our findings indicate that induction of EMT, increased migration and survival depend, in MCF-7 and H460 cells, on the release of FHC control on two pathways, namely the iron/ROS metabolism and CXCR4/CXCL12 axis. Besides constituting a further confirmation of the multifunctional nature of FHC, this data also suggest that the analysis of FHC amount/function might be an important additional tool to predict tumor aggressiveness.