Gene Expression Analysis of The Role MiRNA-148a During Skin and hair follicle development
Abstract
Human skin is the largest organ in the human body with regenerating potential during its entire lifecycle. It protects the internal organs from environmental risk, as well as having a significant role in the repair and healing process via a range of biological mediators that exert local or systemic effects. The skin comprises three layers, the dermis, the epidermis, and the hypodermis. There are several molecular pathways, growth factors, genes, and proteins which have an essential role in skin development and hair follicle, such as, FGF, BMP, hedgehog, Notch and wingless-type integration site (Wnt). The Wnt/β-catenin signalling pathway plays a crucial role in growth, proliferation and hair follicle development in addition to the regulation of the activity of embryonic stem cells. BMP signalling plays a significant role in the regulation of skin development and is essential in embryogenesis. The fate of placodes formation in surrounding cells is regulated by BMP signalling activity. Shh plays a significant role in the regulation of dermal PDGFRA expression and several Shh downstream effectors, such as TGF-β, Wnt5a and PDGFRA, are reported within the dermal condensates. Skin appendages, such as hair follicles, sebaceous glands, sweat glands, and nails are derived from the ectodermal layer, which functions as an external barrier to defend the internal organs from internal and external environmental dangers. MicroRNAs (miRNAs) are short length non-coding RNAs made up of approximately 22 nucleotides developed from endogenous primary transcripts with a local hairpin structure through enzymatic cleavage. They have a vital role in the proliferation of stem cells and the formation of keratinocytes. Also, miRNAs play an essential role in skin function by maintaining stemness in the skin and other stratified epithelial tissues. There is increased expression of miRNAs in the keratinocyte in psoriasis and chronic venous ulcers. This research aimed to evaluate and investigate the role of miRNA-148a and cytokeratin 1 (K1) in calcium-induced keratinocyte differentiation in vitro. Several methods and techniques were used to investigate the gene expression of both miRNA-148a and K1 post in HaCaT cells cultured in various calcium concentrations, including skin and hair follicle histology, RNA extraction, gel electrophoresis, (cDNA) synthesis and RT-qPCR. The results revealed that K1 expression in HaCaT cells cultured in 1.8 mM calcium was higher than in low calcium (0.05 mM) concentrations, one-fold by 24 hours post-differentiation, increasing to six-fold by 48 hours, decreasing thereafter to one-fold to equate with low calcium (0.05 mM) treatment, the stander control. In contrast, the miRNA 148a gene expression in HaCaT cells cultured for 24 hours in
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high Ca+2 (1.8 mM) was less than one-fold, increasing to around 1300-fold by 48 hours, then, decreasing to 40-fold at 72 hours. Both one-way ANOVA and n unpaired t-tests were conducted to compare miRNA expression in low and high calcium conditions. These findings suggest that miRNA-148a might be helpful in therapy for several diseases of the skin and their appendages, as well as serving as a predictive and prognostic marker in the diagnosis of skin cancer. However, the target genes of upregulated miRNA-148a in keratinocyte differentiation have not been identified, therefore further investigations are required to determine the role of miRNA- 148 in keratinocyte differentiation. Keywords: keratinocyte differentiation, calcium, wingless-type integration site (Wnt), FGF, BMP, hedgehog, skin appendages, HaCaT cell line, miRNA-148a, cytokeratin 1