089 MiR-30a is an aged-related microRNA that impairs differentiation and induces apoptosis in human epidermis

Abstract

ABCA12 is known to be critical for skin barrier integrity. Mutations in this gene cause the most severe form of Autosomal Recessive Congenital Ichthyosis, Harlequin Ichthyosis (HI). HI patients have marked hyperkeratosis at birth with fissuring, leading to life-threatening complications due to increased risk of infection, trans-epidermal water and heat loss. The aim of this study was to identify essential pathways involved in the pathomechanisms of Harlequin Ichthyosis, responsible for aberrant epidermal differentiation. We performed RNA-seq on calcium induced primary keratinocytes with siRNA knockdown of ABCA12 and identified 118 genes significantly down-regulated and 36 genes significantly up-regulated (FDR < 0.05). Functional annotation clustering analysis showed changes in epidermal differentiation, fatty acid metabolism, cytokine and interferon signaling. The suppressor of cytokine signaling 3 (SOCS3), a negative feedback regulator of the JAK-STAT signaling pathway, was 2.5 fold downregulated whereas Interleukin-1 (IL1A and IL1B) were 2 fold increased. To investigate these findings further we engineered an ABCA12 CRISPR-Cas9 knockout keratinocyte cell line and compared this with a HI patient-derived cell line and wild type controls. Alterations in differentiation and lipid profile in the HI OT models were observed, recapitulating the HI epidermis phenotype. We found that phospho-STAT1 (Y701) was strongly upregulated in the HI model compared to control. In HI patient skin, the STAT1 expression pattern was altered compared to control skin. The secretion of IL-1a was increased in the HI model compared to control. Both STAT1 and IL-1 regulate the Nitric Oxide (NO) pathway upregulating transcription of inducible NO synthase (iNOS), which we found to be significantly upregulated in HI skin. These data provide insights into the pathogenesis of HI suggest that the NO signaling pathway may be a possible therapeutic target in this disorder. 089MiR-30a is an aged-related microRNA that impairs differentiation and induces apoptosis in human epidermis The age-related alterations of epidermis are essentially characterized by a defect in the tissue renewal caused by a reduced keratinocytes proliferation and by a disturbance of their differentiation. However, the mechanisms disturbing epidermal homeostasis during aging remain poorly understood. To go further into this question, we focused on microRNAs (miRNAs), a class of non-coding RNAs known to play a key role in the regulation of epidermal homeostasis. To identify age-related microRNAs, we performed an exhaustive miRNAs microarray expression screen in human primary keratinocytes from young or elderly peoples. This screening revealed many microRNAs modulated by aging including miR-30a, exhibiting both strands overexpressed in aged cells and tissue. We also observed that miR-30a-3p and -5p were significantly induced in a reconstructed skin model mimicking chronological aging. We then constructed a lentiviral vector allowing its inducible and stable overexpression in primary...