MIR-206-3P Dynamics During Mouse Skin Development And Its Potential Roles

The cutaneous nerve terminals grow transiently to the skin surface and contact directly with outside amniotic fluids (AF) before retracting subepidermally during embryonic stages. However, the mechanism behind the plasticity forming of cutaneous nerve during embryonic development remains elucidated. This study aims to explore the potential roles of microRNAs (miRNA) in this context by starting from determinating the differential expressions of miRNAs in AF.Firstly, AF specimens of male fetal mouse on13.5dpc (day postcoitum),15.5dpc and17.5dpc were collected by amniocentesis. These AFs were centrifugated at16000×g for10minutes to remove any cells and debris. Then, RNAs were extracted from the AF supernatant by TRIzol(?) reagent. A miRNAs microarray coated with LNA’M probes was used to profile the miRNAs expression of AF. Expression data on the three embryonic time points were analyzed by k-medians clustering. After sorting these clustered data on expression intensity, miR-206-3p a candidate probably related to skin function was selected as a miRNA of interested.Secondly, a stem-loop RT-qPCR was established for quantitating miR-206-3p. In order to develop appropriate method to fix fetus in advance, two fixatives [methanol/DMSO (4:1) and paraformaldehyde] were applied for embryo (12.5dpc) fixation and two preservatives (methanol and30%sucrose) were used for the fixed embryo preservation. After storage for one month, the skin, skeletal muscle and brain tissues were dissected from the fixed and unembedded embryos. Total RNAs were extracted by TRIzol(?) reagent, and then were subjected to amplify Actb, Hprt, Gapdh, Rnu6, Snord68and miR-206-3p by RT-qPCR. Embryos fixed in methanol/DMSO and preserved in100%methanol at-20℃were able to yield at least349bp amplifiable RNA. Using Snord68and/or Rnu6as internal control, we found that the miR-206-3p expression level in skin was about one quarter of its highest level in the skeletal muscle.It was reported that miR-206-3p is involved in the airway smooth muscle innervation through its post-transcriptional silencing on brain-derived neurotrophic factor (BDNF). We supposed that the cutaneous innervation may also adopt this regulation strategy. The full-thickness back skins from13.5dpc,15.5dpc,17.5dpc,1dpp (day postpartum),4dpp and16weeks mice were collected, respectively. The expression levels of miR-206-3p and Bdnf mRNA were measured by RT-qPCR, while BDNF protein by western blotting. The tissue distributions of miR-206-3p and BDNF were profiled by in situ hybridization (ISH) and immunohistochemistry (IHC), respectively. The expression level of miR-206-3p increases gradually till17.5dpc, and recedes subsequently to a similar level to13.5dpc by adult stage. During this period, BDNF protein expresses in the opposite direction to miR-206-3p while Bdnf mRNA remains stable after17.5dpc. ISH results showed that the miR-206-3p distribution is regionalized during embryonic period and mainly locates in the suprabasal layer and periphery of hair follicles, which is adjacent to BDNF distributions in a nonoverlapping manner. These results suggest that miR-206-3p may be involved in mouse skin neurodevelopment through its post-transcriptional regulation of Bdnf.The distribution areas of miR-206-3p during skin development are also the place where epidermis begins to cornification and epidermis contacts with dermis. This suggests miR-206-3p may relate to specialization on functions of keratinocyte (KC). To further understand miR-206-3p expressed in the epidermis and dermis tissue, the major constitutional cell of these two tissues—KC and dermal fibroblasts (DF) were dissected from1dpp neonatal mouse and primary cultured, respectively. The expression level of miR-206-3p in KC was lower than that in DF (t=-16.618, P=0.000, n=6). The expression levels of Bdnf mRNA were similar between KC and DF (t=-2.632, P=0.058, n=6). While the BDNF protein expressions in KC were higher than that in DF (pro-BDNF:t=-9.222, P=0.001, n=6; BDNF:t=4.841, P=0.008, n=6). To determine whether miR-206-3p dynamics is dependent on the epidermal differentiation, in vitro calcium-induced differentiation of primary KC was performed. However, no significant differences were found in the expression of either miR-206-3p or Bdnf during differentiation (miR-206-3p:t=-1.721, P=0.227; Bdnf mRNA:t=-2.640, P=0.118; mature BDNF:t=-2.815, P=0.106; pro-BDNF:t=0.135, P=0.905; n=3), indicating that miR-206-3p is not related to KC differentiation.Lastly, a full-thickness skin wound model eliminating influence from appendage during wound healing was established by cutting back skin of5mm in diameter. Then, the healing tissues on the6th,9th and12th day after wounding were collected, respectively. In this course, miR-206-3p little expressed in healing tissues, which determined by qPCR and ISH. While the mRNA and protein expressions of Bdnf were down-regulated at early stage and up-regulated at late stage. The miR-206-3p expression during skin embryonic stage and healing course are apparently different, but share some features. Through the post-transcriptional suppression of Bdnf, miR-206-3p performs its mission in neuronal development and cutaneous innervation during embryonic stage, and plays a role on proper regulation in neuronal repairing during wound healing.Our findings on miR-206-3p involved in skin development will renew current understandings of this muscle specific miRNA, which add new proofs for miRNAs functioning in a tissue specific manner. The new role of miR-206-3p in regulating BDNF during cutaneous innervation could help to reveal the neuronal function established in skin. It makes the possibility to explore the mechanism of cutaneous innervation from a miRNA’s view, and suggests a new mechanism that can be targeted for intervention.