The AR/miR-221/IGF-1 pathway mediates the pathogenesis of androgenetic alopecia

Androgenetic alopecia (AGA) affects more than half of the adult population worldwide and is primarily caused by the binding of dihydrotestosterone (DHT) to androgen receptors (AR). However, the mechanisms by which AR affects hair follicles remain unclear. In our study, we found that miR-221 significantly suppressed hair growth and the proliferation of dermal papilla cells (DPCs) and dermal sheath cells (DSCs) in AGA patients. Interestingly, miR-221 and AR were mainly co-located in the same part of the hair follicle. Mechanistic analysis revealed that AR directly promoted the transcription of miR-221, which in turn suppressed IGF-1 expression, leading to the inactivation of the MAPK pathway in DPCs and the PI3K/AKT pathway in DSCs. In AGA patients, miR-221 expression was positively correlated with AR expression and negatively correlated with IGF-1 expression. Our findings indicate that miR-221, as a direct target of AR, plays a crucial role in the pathogenesis of AGA, making it a novel biomarker and potential therapeutic target for treating AGA.

manufacturer's protocol. To quantify mRNA expression, polyadenylated total RNA underwent reverse transcription using PrimeScript™ RT Master Mix (TaKaRa, Dalian, China). An SYBR® Premix Ex TaqTM II kit (TaKaRa, Dalian, China) was utilized for real-time PCR on a Roche LightCycler480 system. To quantify miR-221 expression, polyadenylated total RNA underwent reverse transcription using an NCode miRNA First-Strand cDNA Synthesis kit (Invitrogen). Real-time PCR was conducted using an SYBR Green PCR master mix (Applied Biosystems; Foster City, Calif, USA) on a Roche LightCycler480 system. GAPDH or U6 snRNA were used as internal controls. All samples were normalized to these controls, and fold changes were calculated through relative quantification (2-ΔΔCT) as is standard. The specific primers used are detailed and can be found in Supplementary Table 1.

Organ culture and treatment
Anagen hair follicles were cultured and measured as previously described [4]. Briefly, the hair follicles were cultured in Williams E medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10 ng/ml hydrocortisone, 2 mM L-glutamine, 10 mg/ml insulin, and 100 U/ml streptomycin for 7 days in 24-well dishes. The hair follicles were photographed using a Leitz Labovert FS inverted microscope (Wetzlar, Germany) and the length was measured every 24 hours for 8 days. The stage of hair cycle was classified according to a previous study [5].
For IGF-1 or DHT treatment, 1000 ng/mL IGF-1 (Abcam, London, UK) or 8000 nM DHT (Solarbio, Beijing, China) was added to the cultured cells every two days. For transfection, hair follicles were transfected with 200 μM miRNAs using Lipofectamine

In vivo experiment
All animals used in this study were purchased from the Experimental Animal Centre at Southern Medical University (Guangzhou, China). After shaving, 3-week-old littermate C57BL/6 mice received intradermal injections of miR-221 angomir, antagomir, angomir control, or antagomir control (3mg/kg) every 5 days, as previously described [6][7][8]. we used a 3*2 grid pattern to inject at 6 points on the dorsal skin of mice, with a volume of 100μl per injection point. The animals were examined and photographed every 6 days and sacrificed on day 18 for histology and immunofluorescence analysis.
The dorsal skin of the sacrificed mice was excised and fixed in 4% paraformaldehyde at 4 °C for a maximum of 1 week. For histological analysis, the dorsal skin samples were embedded in paraffin blocks and 3 µm thick sections were prepared according to longitudinal sections of hair follicles, followed by hematoxylin and eosin (HE) staining.
Digital photomicrographs of representative areas were obtained. Hair bulb diameter was determined using Image-Pro Plus software.

Cell proliferation assays
Cell proliferation assays were conducted using the Cell Counting Kit 8 (CCK-8) (Dojindo; Kumamoto, Japan). Cells were seeded at a density of 1 × 10 4 cells per well in 96-well plates and cultured in the growth medium. At the indicated time points, the number of cells was measured in triplicate wells by detecting the absorbance at 450 nm.

miRNA in situ hybridization
In situ hybridization assays for mature hsa-miR-221 in hair follicle tissue sections were conducted using digoxin-labeled oligonucleotide probes. The specific hsa-miR-221 probe (5'-GAAACCCAGCAGACAATGTAGCT-3') and negative control scrambled-miR probe (5'-TTCACAATGCGTTATCGGATGT-3') (Sangon Biotech, Shanghai, China) were utilized. The miRNA in situ hybridization was performed in accordance with previously reported procedures [9]. Optical signals were visualized using an Olympus BX63 microscope (Tokyo, Japan). Specifically, after incubation with 10 mM EdU for 2 hours, cells were fixed using 4% paraformaldehyde, permeabilized with 0.3% Triton X-100, and stained with Apollo fluorescent dyes. 5 μg/ml of DAPI was utilized to stain the cell nuclei for 10 minutes.

Cell cycle analysis and EdU incorporation assay
The number of EdU-positive cells was assessed under a fluorescent microscope in five random fields. All assays were repeated independently three times.