miR-370-3p Inhibited the Proliferation of Sheep Dermal Papilla Cells by Inhibiting the Expression of SMAD4.

Abstract

The proliferation and maturation of hair follicles in follicular papilla cells are predominantly governed by miRNAs, which significantly influence the cell cycle, apoptosis, and proliferation. miR-370-3p has been associated with several biological processes and targets SMAD4, a crucial component in hair follicle development. Tissue expression profiling revealed significant differences in miR-370-3p levels between skin tissues of the two sheep breeds in January and October, as well as between tissues of the Xinji fine-wool sheep and Small-tail Han sheep. SMAD4 exhibited significant differences in tissue-specific expression in the heart, spleen, skin, lungs, and muscles from Xinji fine-wool sheep and Small-tail Han sheep. Bioinformatics analysis and dual-luciferase reporter assays validated the regulatory interaction between miR-370-3p and SMAD4. CCK-8 experiments demonstrated that miR-370-3p's targeting of SMAD4 suppressed cell growth. Cell cycle analysis demonstrated that miR-370-3p's targeting of SMAD4 influenced the cell cycle. Annexin V-FITC/PI dual labeling demonstrated that miR-370-3p's targeting of SMAD4 promoted cell apoptosis. RT-qPCR data demonstrated that miR-370-3p's targeting of SMAD4 elevated the expression of JUN, c-MYC, and TCF7L2 while suppressing β-catenin expression. Western blot (WB) analysis demonstrated that miR-370-3p targeting of SMAD4 significantly promoted c-MYC expression while inhibiting CCND1, CCND2, and β-catenin expression. miR-370-3p and SMAD4 exhibit spatiotemporal expression differences in sheep skin tissues, with widespread expression across various tissues. Furthermore, it confirmed that miR-370-3p targets SMAD4 to inhibit follicular papilla cell proliferation, promote apoptosis, and influence the cell cycle.