MicroRNAs regulate granulosa cells apoptosis and follicular development — A review

Objective MicroRNAs (miRNAs) are the most abundant small RNAs. Approximately 2,000 annotated miRNAs genes have been found to be differentially expressed in ovarian follicles during the follicular development (FD). Many miRNAs exert their regulatory effects on the apoptosis of follicular granulosa cells (FGCs) and FD. However, accurate roles and mechanism of miRNAs regulating apoptosis of FGCs remain undetermined. Methods In this review, we summarized the regulatory role of each miRNA or miRNA cluster on FGCs apoptosis and FD on the bases of 41 academic articles retrieved from PubMed and web of science and other databases. Results Total of 30 miRNAs and 4 miRNAs clusters in 41 articles were reviewed and summarized in the present article. Twenty nine documents indicated explicitly that 24 miRNAs and miRNAs clusters in 29 articles promoted or induced FGCs apoptosis through their distinctive target genes. The remaining 10 miRNAs and miRNAs of 12 articles inhibited FGCs apoptosis. MiRNAs exerted modulation actions by at least 77 signal pathways during FGCs apoptosis and FD. Conclusion We concluded that miRNAs or miRNAs clusters could modulate the apoptosis of GCs (including follicular GCs, mural GCs and cumulus cells) by targeting their specific genes. A great majority of miRNAs show a promoting role on apoptosis of FGCs in mammals. But the accurate mechanism of miRNAs and miRNA clusters has not been well understood. It is necessary to ascertain clearly the role and mechanism of each miRNA or miRNA cluster in the future. Understanding precise functions and mechanisms of miRNAs in FGCs apoptosis and FD will be beneficial in developing new diagnostic and treatment strategies for treating infertility and ovarian diseases in humans and animals.


INTRODUCTION
The first microRNA (miRNA) was discovered in the Caenorhabditis elegans by Ambros and Ruvkun in 1993 [1]. Later, a lot of miRNAs were discovered in human and animals [2]. The miRNAs are originally transcribed from coding genes which occupy 1% to 3% of the genome. Currently, approximately 2,000 annotated miRNAs have been reported in humans [3,4]. The miRNAs regulate about 30% of proteincoding genes in mammals since the different miRNAs may target the same mRNAs [5]. Nucleotides sizes of miRNAs are dif ferentially reported in the mammals, including 19 to 22 nucleotides [6], 20 to 24 nucleotides [6,7], 21 to 23 nucleotide [4], and even 21 to 26 molecules [8]. Consequently, the precise numbers of nucleotides of miRNAs remain undetermined [9,10].
Roughly, 52% of human miRNAs are localized within the intergenic regions, 40% are located within intronic regions, and the rest 8% are situated within exons [11]. In mammals, most miRNAs regulate gene expression via combining the 3′untranslated region (UTR) and the specific sequences of target mRNAs, causing repres sion of translation of target mRNAs [1214]. One miRNA may target hundreds of different mRNAs. However, the regulatory mechanism of every miRNA remains unclearly understood [12].
Follicular granulosa cells (FGCs) play a key role in nourish ing oocytes through secreting growth factors and hormones and regulating development of oocytes [15]. It has been well known that miRNAs exert the vital functions in FGCs apop tosis and follicular development (FD) [8,16,17]. The functions of specific miRNAs are implicated in different aspects of FGCs processes of the mammals, such as proliferation [18], differ entiation [19], and cumulus expansion [20].
Previous studies aimed to determine the roles of miRNAs on the FD of mammals using various approaches, includ ing conditional knockout of miRNA biogenesis genes, high throughput sequencing technologies in various animal models. Nowadays, it has been well known that miRNAs exert a sig nificant role in FD and oocyte development of mammals [8,21].
However, so far the accurate effects and regulatory mech anism of different miRNAs regulating apoptosis of granulosa cells (GCs) and FD have still remained unclear, especially their target genes and signaling pathways [20,22]. The present review aimed to comprehensively elaborate the research ad vances on miRNAs for modulating apoptosis of FGCs and FD in humans and animals so as to seek new diagnostic and treatment scheme for infertility and ovarian diseases.

miRNAs MODULATE APOPTOSIS OF FOLLICULAR GRANULOSA CELLS
The miRNAs regulate the function of FGCs via altering ex pression levels of target genes [7,23]. The microRNA (miR) let7 family is highly conserved in sequences across animal species. MiRlet7 family is differentially expressed during follicular atresia [24]. Expression levels of miR-let-7a, let-7b, let-7c, and let-7i genes were reduced in early and progressed atretic follicles as compared to those in healthy follicles [25,26]. The miRlet7gmediated suppression of mitogenactivated protein kinase kinase kinase 1 (MAP3K1) resulted in the ex pression and dephosphorylation of the transcription factor fork head O1 (FOXO1) which induced FGCs apoptosis [27]. Overexpression of miRlet7g increased the apoptosis rate of the mouse FGCs [26] and FOXO1 expression in FGCs, and then resulted in nuclear accumulation of dephosphorylated FOXO1. Additionally, the expression levels of the apoptosis associated genes including Caspase 3, BCL2Associated X (BAX), and BES1interacting Myclike protein (BIM) were significantly upregulated after miRlet7g mimic was trans fected into porcine FGCs. But the antiapoptotic genes Bcell lymphoma2 (Bcl-2) and myeloid cell leukemia1 were sig nificantly downregulated [26]. Briefly, the miRlet7 family exerted a potential in the regulation of FGCs apoptosis.
MiR21 is one of three highly luteinizing hormone (LH) induced miRNAs in murine FGCs [14]. It acts as an anti apoptotic factor in GCs. A loss of miR21 in vivo leads to a reduction of ovulation rates [28]. MiR21 blocks the apoptosis of murine FGCs [14,29]. Several miR21 target transcripts have been identified to explain its antiapoptotic effect, in cluding programmed cell death 4, phosphatase and tensin homologue [29,30].
The levels of the primary transcript of miR21 (primiR21) and mature miR21 were obviously increased in the cumulus oocyte complexes (COCs) over the maturation period. The primiR21 expression was remarkably decreased in COCs treated with a signal transducer and activator of transcrip tion 3 pathway inhibitor, and cumulus expansion may be prevented. Inihibition of PrimiR21 expression directly in fluenced miR21 expression in bovine oocytes and cumulus cells (CCs) [31]. Upregulating miR21 expression signifi cantly reduced CCs apoptosis. The oocytesecreted factors (OSFs) upregulated miR21 expression and suppressed FCCs apoptosis by activating the PI3K/Akt signal [29]. It is known that oocytes and CCs are more resistant to apoptosis than other compartments of the antral follicle. However, little is known about the intracellular mechanisms by which OSFs render FCCs resistant to apoptosis [29,32].
MiR146a is implicated in ovarian cancer development by suppressing the expression of antiapoptotic genes, such as X linked inhibitor of apoptosis protein, Bcl2like protein 2, and baculoviral IAP repeat containing 5 [33]. The downregulation of miR146a inhibited apoptosis of FGCs by simultaneously targeting interleukin1 receptorassociated kinase (IRAK1) [34]. A recent study demonstrated that miR126 inhibited FSH receptor (a direct target gene) expression and increased the apoptosis rate of porcine FGCs [35]. However, the cell apoptosis rate was dramatically reduced when miR1413p was overexpressed in rat FGCs [36].
The miR144 was differentially expressed in the porcine preovulatory follicles. The miR144 regulated FGCs apopto sis and affected follicular atresia [39]. Additionally, miR224 was involved in the mouse FGCs proliferation via targeting SMAD4 [33]. Another study indicated that miR1275 was expressed during the porcine follicular atresia. The miR1275 can promote early apoptosis of porcine FGCs and the initia tion of follicular atresia (FA) by inhibiting estradiol release and expression of liver receptor homolog (LRH)1 that was bound to the cytochrome P450, family 19, subfamily A, poly peptide 1 promoter and increased its activity. Additionally, miR1275 attenuated LRH-1 expression by directly binding to its 3′ UTR [40].
Expression of miR130b was altered during oocyte matu ration by directly targeting SMAD5 and mitogen and stress activated protein kinase 1 which were identified as target genes of miR130b. Overexpression of miR130b increased the pro liferation of mural GCs and CCs. But, inhibition of miR130b expression during in vitro maturation (IVM) of oocytes de creased the first polar body extrusion and the mitochondrial activity. Such, functional modulation of miR130b affected the proliferation and survival of GC and CC as well as oocyte maturation [47].
Overall together, studies on cell communication, extracel lular matrix and signaling pathways have demonstrated the differential expressions of miRNAs have relevance with physi ological functions of CCs and mural GCs [45].

MiRNAs REGULATE DEVELOPMENT OF OVARIAN FOLLICLES
Both FD and oocyte maturation are completed in the ovaries of female mammals. A highly complicated, spontaneous death phenomenon that is called as atresia takes place during the FD and maturation in the mammals. Follicular atresia is re sulted from the apoptosis of GCs surrounding oocytes [49]. In mammals, less than 1% of ovarian follicles will eventually ovulate. More than 99% of ovarian follicles are disappeared as a result of atresia, which affects all stages of follicular growth and development [50].
The FD is mediated by various regulatory factors includ ing many miRNAs [51]. Numerous miRNAs play important roles in follicular atresia and development [49,52]. The miR NAs exert their functions as mediators of these processes via their extensive involvement in posttranscriptional mRNA regulation [53,54]. The miRNAs are differentially expressed during the primordial development [55], luteal development [56] and the whole FD [54].
MiR378 could affect oocyte IVM by inhibiting the ex pansion and altering gene expression of CCs, and adjust in estradiol production by depressing aromatase translation in porcine FGCs. The miR378 decreased IVM rate, sup pressed the expression of genes associated with FD, such as bone morphogenetic protein 15 and growth differentiation factor 9 and also increased apoptosis rate [21] since miR378 targeted to the 3′UTR of aromatase mRNA [57].
In summary, up to date many miRNAs mediate the process of oocyte maturation and folliculogenesis and also regulate follicular atresia through their target genes, thereby modu lating FGCs apoptosis [59]. A large number of miRNAs and miRNA clusters involved in the FD have been documented [6163]. However, accurate roles of miRNAs and miRNA clusters in this process are not clearly understood [8,63]. Understanding the miRNAs roles will elucidate clearly the mechanisms of GC apoptosis, development and atresia of ovarian follicles [64]. A miRNA cluster comprises of more than two miRNAs with similar functions [65]. Currently, it has been reported that the specific miRNA families and clusters are involved in fol licular atresia and development including miR21, miR23a, miR145, miR503, miR224, miR383, miR378, miR132, miR212, the let7 family, miR1792 cluster, miR232724 cluster, miR18396182 cluster, miR1792 cluster and so on [28,64,66,67]. However, it has been undetermined which miRNA cluster(s) are associated with the each stage of FD [61,63,64]. Furthermore, actual roles of these miRNA clusters in the FD, atresia and ovulation remain unclear [8,43,68].

MiRNA CLUSTERS REGULATE FGCs APOPTOSIS AND FOLLICULAR DEVELOPMENT
Fifteen different miRNAs were found during the growth and selection of dominant follicles [69]. Six miRNAs, includ ing miR17, miR18a, miR19a, miR20a, miR19b, and miR 92a, are encoded by a single miR1792 transcript [70], and are expressed and processed together as a cluster [63]. The miR1792 cluster was differentially expressed in GCs from subordinate and dominant follicles at day 19 of the estrous cycle [71]. The overexpression of the miR1792 cluster promoted GC proliferation and reduced the proportion of differenti ated cells. However, miR1792 cluster inhibition resulted in decreased proliferation and increased differentiation in GCs [71].
The miR18396182 cluster (miR183, miR96, and miR 182) is highly conserved [72], it is also abundantly expressed in both luteal cells and bovine FGCs of preovulatory domi nant follicles [20,73]. This miRNAs cluster impacted bFGCs proliferation. The overexpression of miR18396182 promot ed the proliferation of bovine FGCs [20]. This cluster targeted the 3′ UTR of the FOXO1 gene [74], and thus regulated FD and luteal development via exerting effects on cell survival and steroid production. Moreover, it was also reported miR 182 inhibited FGCs apoptosis by targeting SMAD7. However, the actual roles and mechanism of miRNAs remain to be comprehensively investigated in the FGCs apoptosis and fol licular atresia [8,75,76].
The miR232724 cluster comprises the miR23a gene cluster (miR23a, miR27a, and miR242 genes) and the miR 23b cluster (mir23b, mir27b, and mir241 genes) that exert their function via SMAD5. SMAD5 is a direct target of mir 23a and mir27a, which promote GC apoptosis via the Fas FasL pathway [59]. These evidences suggest that miR2327 24 clusters play a role in follicular atresia. On the other hand, expression levels of miR23a27a24, miR222221, and miR 214199a clusters showed an increase until the midluteal phase, but expression decreased in the dominant FGCs dur ing the late follicular phase of the estrous cycle.
Based on the reported information in recent years, the regulatory roles of miRNAs on FGCs are summarized in Table  1. As presented in table 1, in total of 41 academic theses re garding 34 miRNAs and miRNAs clusters that reported the regulatory effects of miRNAs on FGCs apoptosis in mam mals. The documents indicated explicitly that 24 miRNAs and miRNAs clusters in 29 articles promoted or induced FGCs apoptosis through their distinctive target genes. Seven miRNAs inhibited FGCs apoptosis. So far, the regulatory roles of the remaining 9 miRNAs and miRNAs clusters have been undetermined. We could conclude that a majority of miRNAs show promoting role on apoptosis of FGCs in mam mals. But the accurate mechanism of miRNAs and miRNA clusters have been not well understood.

MOLECULAR SIGNALING PATHWAYS WERE SUMMARIZED
The existence of miRNAs was discovered more than 20 years ago, and since then considerable achievements have been made in understanding the molecular mechanisms in the apoptosis, proliferation and development of follicular cells [78]. MiRNAs can combine with complementary sequences in the 5′UTR [52] or 3′UTR [21] of target mRNAs, there fore degrading the mRNA or repressing translation.

1719
Gong et al (2020) Asian-Australas J Anim Sci 33:1714-1724 and Bcl2 (Figure 1). Moreover, the SMAD played an important role in regulat ing FD [51,81]. SMAD proteins can transduce the TGFβ family signals at the cell surface into gene regulation in the nucleus. The miR23a and miR27a targeted SMAD5 and regulated apoptosis in human GCs via the FasLFas pathway [59].
The functional networks play critical roles in the FD which contribute to the profound exploration on miRNAs roles. However, the association with downstream apoptosis genes and proteins remains still unclear [85,86]. The exact signal pathways in which the miRNAs exert need to be investigated in the future [40].

CONCLUSIONS AND PERSPECTIVES
MiRNAs are involved in physiological and developmental processes by posttranscriptionally inhibiting gene expres sion. In this review of 41 academic theses, we summarize the current advances in the regulatory roles of miRNAs and miRNA clusters on the FGCs apoptosis and FD in the mam mals. Total of 30 miRNAs and 4 miRNAs clusters were reported in all articles. The documents indicated explicitly that 24 miRNAs and miRNAs clusters in 29 articles promoted or induced FGCs apoptosis through their distinctive target genes. The remaining 12 papers reported that 10 miRNAs and miRNAs clusters inhibited FGCs apoptosis. We could conclude that miRNAs or miRNAs clusters could modulate the apoptosis of GCs (including follicular GCs, mural GCs, and cumulus cells) by targeting its specific genes through the different signal pathway. A majority of miRNAs show promoting role on apoptosis of FGCs in mammals. But the accurate mechanism of miRNAs and miRNA clusters is not well understood [8,43,64]. The current results in the published documewnts are still not to clearly eaplain the distinctive effects of each miRNAs or miRNA cluster on FGCs apoptosis and FD in mamnals. It is extremely necessary to ascertain clearly the role and mechanism of each miRNA or miRNA cluster in the future. Understanding comprehensively mech anism of miRNA action may enhance the development of new tools to study miRNAs functions and inspire new di agnostic and treatment strategy or scheme for infertility [87], ovarian disorders and ovarian diseases associated to miRNA high expression or insufficiency [88], such as fol licular infertility and ovarian cancer [89].

AUTHOR CONTRIBUTIONS
Professor Zhuandi Gong proposed the subject and designed the article. Dr. Xiaoyun Shen retrieved the references. Miss Juan Yang and Luju Lai analyzed the data of documents. Miss Bai Shengju drew the tables. Professor Suocheng Wei designed the article and wrote the manuscript.

CONFLICT OF INTEREST
We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manu script.