In animal husbandry, poultry productivity is closely associated with improving reproductive performance. Follicle development and growth are important factors affecting chicken reproductive potential. One mature follicle ovulation daily confers the high productivity of chickens [1]. Chicken follicle maturation follows a strict hierarchical sequence. Only a small number of primordial follicles mature from small yellow follicles (SYFs; 6–8 mm in diameter), and only one dominant follicle is chosen per ovulation [2]. The particular mechanisms of follicle selection and development remain elusive. However, granulosa cells (GCs) are assumed to play a vital function in this process [3]. GCs are responsible for mature oocyte production, ovulation, and secretion of steroid hormones such as progesterone (P4) [4], providing a critical microenvironment for follicle growth and maintaining endocrine system homeostasis [4,5]. Previous studies have shown that GCs promoted follicle survival and growth while inhibiting cell apoptosis by stimulating cell proliferation and secreting hormones [6]. GC proliferation and hormone secretion are closely associated with follicle growth and development. Therefore, understanding the mechanisms influencing GC proliferation and hormone secretion is critical.

MicroRNAs (miRNAs) are endogenous 22–24 nucleotide small non-coding single-stranded RNAs that negatively post-transcriptionally regulate gene expression [7]. MiRNA functions have previously been implicated in various physiological processes, such as cell proliferation, apoptosis, and GC function [8]. For example, miRNA 130a-3p reduces estradiol and P4 release by targeting prostate transmembrane protein androgen-induced 1 (PMEPA1) in goat GCs [9]. Similarly, miRNA 181a-5p inhibits goose GC viability [10]. Recent studies have shown that miRNA 140 (miR-140) influences cell proliferation, differentiation, and migration [11,12]. Knocking-out of miR-140 in mice resulted in a considerable increase in GC apoptosis and a decrease in GC proliferation [13]. In addition, miRNA 140-3p (miR-140-3p) promotes adult avian myoblast proliferation and regulates the myoblast cell cycle [14]. However, miR-140-3p's mechanism in chicken GC proliferation and steroid hormone synthesis remains unknown.

Therefore, this study explored miR-140-3p's functions in chicken GCs, cell proliferation, steroid hormone synthesis, and apoptosis and performed target gene prediction to understand better how it influences GC proliferation and steroid hormone synthesis.