Impact of non coding RNAs on skin homeostasis

Non coding RNAs have recently gained increasing attention because of their crucial roles in gene regulatory processes. In the past, functional studies, using human epidermal keratinocytes as a model system, have revealed their role in controlling normal epithelial homeostasis, including cellular senescence, proliferation and differentiation. Ageing of human skin is associated with phenotypic changes in the cutaneous cells; the major functional markers of ageing occur as consequences of dermal and epidermal cell senescence. Senescence encompasses growth arrest during which cells remain metabolically active but acquire a typical enlarged, vacuolar and flattened morphology. Another aspect of skin homeostasis is also played by terminal keratinocytes differentiation. This is an important mechanism to continuously regenerate the barrier against the environment, which excludes foreign substances and organisms and prevents the loss of vital fluids. During epidermal differentiation, keratinocytes lose proliferative potential and move in the basal cell layer of the epidermis through the granular layer where the cornified envelope is formed. Previous studies carried on in the laboratory, identified miRNAs as important regulator of keratinocyte differentiation and senescence. Among them, miR-191 was identified. MiR- 191 over-expression is sufficient per se to induce senescence, as evaluated by induction of several senescence-associated markers. We show that SATB1 and CDK6 3’UTRs are two miR-191 direct targets involved in this pathway. Cdk6 and Satb1 protein levels decrease during keratinocytes replicative senescence and their silencing by siRNA is able to induce a G1 block in cell cycle, accompanied by an increase in senescence-associated markers. Another class of non-coding RNAs (ncRNAs), Transcribed-ultraconserved regions (T-UCRs), is also strongly involved in controlling keratinocyte differentiation and proliferation. T-UCRs are transcribed from regions that exhibit extremely high conservation between the orthologous regions of human, rat and mouse genomes. In the human genomes, T-UCRs are found to overlap with exons of genes involved in RNA splicing or are located within host gene introns or in close proximity of genes that are involved in transcription and development regulation. While ultra-conserved sequences appear to act as regulators of gene expression during development, very little is currently understood about the mechanism by which they act. Here, I focused my attention on T-UC 291, which is up-regulated during human epidermal keratinocyte differentiation. T-UC 291 is a part of an intron of the host protein-coding gene, C10orf11. I showed that this T-UC is predominantly nuclear and I provide evidences that its down-regulation in keratinocytes is sufficient to induce an increase of proliferation rate and a delay in differentiation. My preliminary data suggest that T-UC 291 has an important role in the regulation of cell proliferation and differentiation and probably it acts as transcriptional enhancer to regulate the expression of genes like ZSWMI8 and ZMIZ, localized in proximity of T-UC 291. The data presented in this thesis demonstrated that nc-RNAs (miRNAs and T-UCRs) are important mediators of proliferation, differentiation and cellular senescence in keratinocytes. Studying these pathways may be relevant for maintaining tissue homeostasis and for preventing skin pathologies, such us psoriasis, hyperkeratosis and premature ageing, in which proliferation/differentiation balance is altered.