Role of MEG3-miRNAs in immune-inflammatory regulation in age-dependent wool follicle remodeling in Tan sheep

The “nine-curve” crimped wool is a key economic trait of Tan sheep, which peaks at 35 days of age and disappears in adulthood. However, the regulatory mechanisms underlying this age-dependent trait remain unclear. This study aimed to decipher these mechanisms by analyzing the skin of Tan sheep across critical developmental stages. We integrated small RNA sequencing, mRNA sequencing, and proteomics, combined with phenotypic observation and histological analysis, to examine the skin tissues of Tan sheep at three critical developmental stages (embryonic day 120, trait initiation stage; 35 days of age, trait peak stage; 24 months of age, trait disappearance stage). Phenotypic and histological results confirmed that this crimped trait depends on medullated fibers derived from primary follicles; with individual aging, the ratio of secondary to primary follicles increases, and primary follicles degenerate in adulthood, ultimately leading to the loss of the crimped trait. Transcriptomic analysis showed that the PD-1/PD-L1 immune checkpoint pathway is the core hub mediating “immune-follicle” crosstalk. Proteomic results revealed a “transcriptional reserve-translational delay” characteristic in keratin genes such as KRT5 and KRT71, confirming that the phenotype at 35 days of age is a continuation of the developmental program initiated at embryonic day 120. Small RNA analysis revealed a "miRNA storm" that peaks at embryonic day 120 (41 miRNAs in total, 40 of which are derived from the MEG3 (GTL2)-miRNA cluster on chromosome 18), and the expression level of this storm gradually decreases with age. Multi-omics integration analysis found that these MEG3-miRNAs can target six key genes (HOXC6, RILPL2, RAB10, ARL1, MX1, and ST7) and are significantly enriched in immune-inflammatory related pathways, suggesting that they may participate in the development and remodeling of wool follicles by regulating immune-inflammatory responses, thereby affecting the formation of the crimped trait. This study identifies MEG3-miRNAs as key epigenetic regulators of wool crimping and highlights the role of the immune microenvironment in follicle remodeling. We propose that these MEG3-miRNAs likely inhibit immune activation at embryonic day 120 to support primary follicle development, whereas postnatal reduction of these miRNAs relieves immune suppression, triggering overactivation and primary follicle degeneration. This study provides guidance for Tan sheep breeding, reveals conserved epigenetic‒immune synergy in mammalian skin appendages, and offers a model for studying human age-related hair disorders and cross-species age-dependent traits.