Follicular development is a remarkably orchestrated process, which is divided into several stages, including primordial, primary, secondary, and tertiary follicle [1]. As the functional unit of the ovary, the follicle is composed of an oocyte surrounded by granulosa cells (GCs) and theca cells (TCs) [2], except for primordial and primary follicles. The proliferation and differentiation of GCs are crucial for oocyte maturation and follicular development. GCs can provide ATP and small molecule energy metabolites to oocyte [3]. However, granulosa cell senescence contributes to oocyte dysfunction and the arrest of follicular development [4].

MYB proto-oncogene like 2 (MYBL2/B-MYB), an oncogenic transcription factor, plays a critical regulatory role in cell cycle [5] and cellular senescence [6]. It can facilitate G1/S phase transition via interacting with cyclin D1 [7], and enhance G2/M transition via regulating the expression of CDK1, CCNB1 and CCNA2 genes [8,9]. MYBL2 silencing inhibits proliferation and induces G1-phase cell cycle arrest in the endometrial cancer (EC) cell line [10]. MYBL2 knockdown also decreases the expression of genes and the number of cells in G2/M phase [11,12]. Furthermore, MYBL2 is a direct transcription repressor of the cyclin-dependent kinase inhibitor p16 (INK4α), which has been proposed to play an important role in cellular senescence [13]. Overexpression of MYBL2 decreases G1 block and increases lifespan of human embryonic lung fibroblast cells in vitro, whereas knocking down of MYBL impairs the replicative ability and induces premature senescence. Cyclin-dependent kinase 1 (CDK1) is an archetypical kinase and a central regulator that drives cells through G2 phase and mitosis. Deletion of CDK1 results in cell nonproliferation and entering senescence prematurely [14].

MicroRNA (miRNA) is a short, non-coding, and highly conserved RNA [15], which silences the expression of target genes at the post-transcriptional level mainly by binding to their 3′-untranslated regions (3′-UTR) [16]. It has been well acknowledged that miRNA is extensively involved in the biological processes of GCs, including cell cycle [17,18], proliferation [19], and senescence [20]. miR-143-3p regulates breast cancer cell proliferation and apoptosis by targeting MYBL2 [21]. miR-30b-5p inhibits proliferation and promotes apoptosis of medulloblastoma cells via targeting MYBL2 [22]. miR-495-3p and miR-143-3p co-target CDK1 to inhibit the development of cervical cancer [23]. miR-1271-5p inhibits cell proliferation and enhances the radiosensitivity of hepatocellular carcinoma (HCC) by targeting CDK1 [24].

miR-200 family consists of five members (miR-200a, miR-200b, miR-200c, miR-141 and miR-429). Increasingly, studies have shown that the miR-200 family regulates female animal reproduction through participating in follicle development, estrous cycle, and ovulation. Among them, miR-200b is significantly downregulated in the estrous ovaries compared to the anestrous ovaries of Hu sheep [25], as well as the follicular phase ovaries compared to the luteal phase ovaries of Small Tail Han sheep [26]. The expression of miR-200b is also downregulated in large follicles (LF, >6 mm) compared with small follicles (SM, 2–2.5 mm) in Mongolian sheep [27]. In addition, miR-200b suppresses granulosa cell proliferation in human by directly targeting phosphatase and tensin homolog (PTEN) [28]. Our previous results suggest that miR-200b might regulate the development of ovarian follicular granulosa cells during estrous cycle [26]. Therefore, we give a hypothesis that miR-200b might play an important role in ovine granulosa cell proliferation through some target genes associated with cell cycle. Therefore, we conducted bioinformatic analysis, RNA-seq, plasmid construction and luciferase assays to elucidate the molecular mechanism of miR-200b.