Gene Expression Analysis of MicroRNA-148a During Skin Development

Abstract

Abstract Background: The skin is the largest organ of the body and has many vital roles, including regulation of temperature, protection from infection and prevention of dehydration. The development of embryonic skin growth and regeneration of adult skin, along with hair follicle development, is tightly controlled by many cell signalling pathways and gene silencing and activation by miRNAs. It is known that Ca2+ plays a role in pathways relating to skin development. The full effect of Ca2+ on keratinocyte differentiation, particularly in relation to miRNA and miR-148a will add new understanding to this complex regulatory network. Aim: To investigate the expression and role of miR-148a in calcium-induced keratinocyte differentiation in vitro. Methods: HaCaT cells were cultured and treated with 2.0 mM and 0.05 mM Ca2+ for 24, 48 & 72 hours. RNA extraction with TRIzol was preformed, followed by Nanodrop to quantify RNA, electrophoresis for qualifying and PCR to assay for the housekeeping gene β actin along with K1 and pre-miR148a. Results: Cellular morphology of the HaCaT cells changed in response to high and low Ca2+ treatment to a more differentiated phenotype. This change was correlated with the significant upregulation of the gene expression of K1 and miR-148a after 48 hours incubation with high Ca2+ compared to control cells, as measured by qPCR. Conclusions: miR-148a and K1 expression are increased in response to Ca2+ concentrations in keratinocytes in vitro. Current understanding of miR-148a relate mostly to several cancer types, therefore more research is required to fully understand the complex mechanisms of keratinocyte differentiation and skin development, and the role of cell signalling pathways. miR-148a was also shown to be upregulated in response to high Ca2+ in the present study, the role of which remains unclear. Further experiments should be explored to increase understanding of this particular mechanism in a relation of keratinocyte differentiation.