MicroRNA-148a Controls Epidermal and Hair Follicle Stem/Progenitor Cells by Modulating the Activities of ROCK1 and ELF5.

Skin and hair development is regulated by complex programs of gene activation and silencing and microRNA-dependent modulation of gene expression to maintain normal skin and hair follicle development, homeostasis, and cycling. In this study, we show that miR-148a, through its gene targets, plays an important role in regulating skin homeostasis and hair follicle cycling. RNA and protein analysis of miR-148a and its gene targets were analyzed using a combination of in vitro and in vivo experiments. We show that the expression of miR-148a markedly increases during telogen (bulge and hair germ stem cell compartments). Administration of antisense miR-148a inhibitor into mouse skin during the telogen phases of the postnatal hair cycle results in accelerated anagen development and altered stem cell activity in the skin. We also show that miR-148a can regulate colony-forming abilities of hair follicle bulge stem cells as well as control keratinocyte proliferation/differentiation processes. RNA and protein analysis revealed that miR-148a may control these processes by regulating the expression of Rock1 and Elf5 in vitro and in vivo. These data provide an important foundation for further analyses of miR-148a as a crucial regulator of these genes target in the skin and hair follicles and its importance in maintaining stem/progenitor cell functions during normal tissue homeostasis and regeneration.

Detection of MicroRNAs by In Situ Hybridization in Skin.

MicroRNAs (miRNAs) are a family of small noncoding RNAs (~19-24 nt) playing a key role in the execution of gene expression programs in various cells and tissues. Many technical challenges have been encountered when investigating miRNAs, in particular, determining the spatiotemporal expression pattern of miRNAs in cells and tissues. We describe here a well-established in situ hybridization protocol for the detection and analysis of spatiotemporal expression patterns of miRNAs in skin and its appendages such as the hair follicle in both frozen and paraffin-embedded tissue sections. We describe in detail the different steps that are associated with utilizing in situ hybridization procedure on either frozen or paraffin-embedded tissues for miRNAs localization. Postfixation, tissues are hybridized with LNA double labeled probes with digoxygenin. Detection of hybridized probes is performed by using an alkaline phosphatase coupled antibody against digoxygenin. The final step involves the use of substrates to develop the color of alkaline phosphatase-LNA-probe structure leading to identification of the spatiotemporal location of target miRNAs in target tissue and cells. We also discuss two options for substrate color development in these procedures: (1) NBT/BCIP and (2) BM Purple. This method is a simple and convenient way of determining the spatiotemporal expression pattern of miRNAs, which has been a challenge since their discovery, due to their relatively small size. Knowledge gained from in situ hybridization is crucial for better understanding of the roles of individual miRNA(s) during distinct stages of development in various cells and tissues. These protocols will be beneficial to the wider scientific community.