Follicular development is a highly orchestrated physiological process of ensuring normal reproduction of female animals, which include follicle recruitment, selection and ovulation. Follicular growth during this period requires productive interaction among the oocytes, granulosa cells (GCs) and theca cells [1]. Among follicular cells, proliferation and differentiation of GCs are critical for follicle maturation and ovulation [2]. In these processes, the activities of GCs are regulated by stimulating the synthesis of estradiol (E2), progesterone (P4) by follicle-stimulating hormone (FSH) and Luteinizing Hormone (LH). However, E2 is mainly produced in the pre-hierarchical follicle stage. When entering the pre-ovulation stage, GCs begin to differentiate which is accompanied by the expression of steroidogenic acute regulatory protein (STAR) and cytochrome P450 family 11 subfamily A member (CYP11A1), and produce a large amount of P4 [3,4].

MiRNAs are single-stranded endogenous non-coding small RNAs, which generally include about 21 nt, can regulate gene expression and various physiological processes through complementary interaction with target gene mRNAs [5]. Recently, a large number of studies confirmed that miRNAs are involved in the regulation of ovarian development processes in mammals [[6], [7], [8]] and birds [[9], [10], [11]], including follicular development, atresia, ovulation and degeneration. In this process, miRNAs have been found to play a role by modulating the release of progesterone, androgen and estrogen in the ovary [12]. By analyzing our previous miRNA-seq and RNA-Seq data, we found that miR-10a-5p was differentially expressed between healthy follicles and atretic follicles in chickens [13]. MiR-10a-5p is a member of the miR-10 family, whose function has been elucidated for the first time in diabetic rats and is activated by acarbose and then down-regulated proinflammatory factors [14]. A recent study revealed that miR-10a and miR-10b inhibit proliferation and induce apoptosis in GCs of humans, mice and rat conservatively [15]. Moreover, it has been investigated that circINHA promoted GCs proliferation and inhibited GCs apoptosis by regulating CTGF as ceRNA of miR-10a-5p in pig ovarian follicles [16]. These results indicated that miR-10a-5p might be involved in regulating ovarian follicle growth and ovulation.

Microtubule Associated Protein RP/EB Family Member 1 (MAPRE1), also known as end-binding protein 1 (EB1), is involved in the cell cycle by participating in microtubule dynamic regulation [17]. Initially, MAPRE1 was found to interact with the C-terminal of adenomatous polyposis coli (APC) protein, which demonstrated that c-terminal truncation is associated with sporadic and familial colorectal cancer [[18], [19], [20]]. Further studies showed that the role of APC and MAPRE1 interaction in blocking cancer is mainly to damage the microtubule attachment during mitosis, resulting in the destruction of mitotic spindle and chromosome segregation errors [21]. Recently, some studies have shown that MAPRE1 binds to CDK2, resulting in hyperphosphorylation of CDK2 Thr161 residues in hepatocellular carcinoma cells (HCC), thereby promoting the proliferation of HCC cells and enhancing the occurrence of tumors in vitro. The cell proliferation depends on cell cycle progression through four successive phases: G1, S, G2, and M. The phase transitions are regulated by cyclin-dependent protein kinases (CDKs) that interact with their corresponding cyclin partners [22]. Among them, CDK2 is a member of the CDKs family, which is often used to characterize cell proliferation together with Cyclin D1 (CCND1) and Proliferating Cell Nuclear Antigen (PCNA) [[23], [24], [25], [26]].

In chicken, the specific functions of miR-10a-5p in GCs is still unknown. Thus, in this study, we aim to explore the potential function and the undiscovered regulatory mechanism of miR-10a-5p in chicken GCs proliferation.