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 metab- olism 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 re- constitution 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 quanti ed 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 ndings demonstrate that the molecular mechanism underlying this phenom- enon 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.