Premature ovarian failure (POF) is an ovarian disease caused by many factors and is defined by amenorrhea, high gonadotropic hormone and low estrogen levels in women younger than 40 years old [9]. Granulosa cells play a crucial regulation effect in the growth and development of ovarian follicle. Changes in granulosa cell metabolism and apoptosis rate are closely related to follicular atresia, decreased oocyte quality, ovary ageing and many reproductive endocrinology-related diseases [10]. Xie et al. had found that miRNAs were related to the development of granulosa cells [11]. Yang et al. reported that differentially expressed miRNAs in follicular atresia the plasma of patients with POF might regulate proliferation and apoptosis of granulosa cells by influencing different signalling pathways [12]. The latest studies highlight those miRNAs are essential factors regulating functions of granulosa cell in the physiological and pathological states. Target-specific miRNAs might play an important role in the occurrence and development of POF [13]. According to our previous research, SPPA can significantly inhibit oxidative stress-induced KGN apoptosis, but the molecular mechanisms are unknown. Whether miRNAs are intrinsically involved in regulation is also unknown.
In this study, differentially expressed miRNAs were screened by miRNA-seq. Their functions and target genes enriched functional classification, and signalling pathways were investigated.
A total of 13 differentially expressed miRNAs were identified from the K-vs-H group and H-vs-C group. However, five miRNAs failed to predict corresponding target genes. Specifically, miR-1017, miR-94, and miR-881 are unique and newly reported miRNAs. The remaining seven differentially expressed miRNAs were verified using qRT-PCR. Notably, the expression trends of miR-103a-3p and miR-214 are consistent with miRNA-seq results. However, expression levels of miR-378d, miR-6511b-3p, miR-420, miR-383 and miR-1063 are inconsistent with miRNA-seq results. Such variance might be caused by changes in specific gene expression levels when using different experimental means [14] or due to the low expression level of samples and different sensitivities between qRT-PCR and high-throughput sequencing methods [15]. However, miR-103a-3p and miR-214 play an important role when SPPA inhibits oxidative ovarian damage.
MiR-214 has been shown to regulate cell survival or apoptosis by directly targeting important cell survival-related gene [16]. This miRNA targets Uncoupling Protein 2 (UCP2) and thereby lowers oxidative stress and reactive oxygen species (ROS) levels significantly [17]. Dong et al. studied the protective effect of miRNA-214 on L6 skeletaL yoblast from H2O2-induced apoptosis, reporting that alcohol upregulates the expression of miR-214, both outcomes consistent with data from the current study [18]. Chen et al. demonstrated that miR-214 improves cell survival and inhibit cell apoptosis, and intercepting miR-214 eliminates the protective effect enabling cell survival and anti-apoptosis [19].
Zhou et al. had reported that interfering with miR-103a-3p is able to inhibit LPS-induced hepatocyte apoptosis, inflammation and oxidation reactions [20]. Cheng et al. found that upregulation of miR-103a-3p promoted chondrocyte proliferation of human osteoarthritis (OA) and inhibited cell apoptosis and inflammatory responses [21]. Zhang et al. reported that miR-103a-3p exerted negative influences on the maturation of oocytes by regulating brain-derived neurotrophic factor (BDNF) expression in human follicular fluid [22]. These studies all confirm that miR-103a-3p and miR-214 play an important regulatory role in cell apoptosis and oxidative stress, consistent with the data in the current study.
Hu found that miR-383 was capable of inducing ROS generation and promote cell apoptosis [23]. Relevant studies have shown that miR-383 is associated with reproduction-related miRNA. However, the effects of miR-383 in follicular development have not been considered in detail. Yin et al. had found that miR-383 mainly is expressed in mice's ovarian granulosa cells and oocytes [24]. Li et al. further demonstrated that miR-383 inhibits the proliferation of ovarian granulosa cells in mice by downregulating expressions of cell cycle-related proteins [25]. In this study, sequencing results demonstrated that miR-72 and miR-420 had identical gene sequences and target genes. However, there is no previous studies on the relationship between miR-72 and miR-420, which needs to be further explored and studied.
Additionally, this study deduced the biological functions of 647 target genes from the GO functional annotation and pathway enrichment analysis. Results showed that on the biological processing level, these target genes significantly enrich transcription processes, DNA templates, negative transcriptional regulation of RNA polymerase II promoter, and negative regulation of cell proliferation. On the molecular functional level, they mainly enrich protein binding. Signalling pathway enrichment analysis confirmed that these target genes significantly enrich metabolic pathways, MAPK signalling pathways, lysosome, and axon guidance. In their study of key genes in resveratrol-induced apoptosis in porcine ovarian granulosa cells, Zhang et al. found that the pathways significantly enriched were all associated with hormone secretion and metabolic pathways [26]. Zhang et al. studied miRNA expression spectra in the ovary of rats with insulin resistance polycystic ovary syndrome (PCOS) and found that the predicted target genes correlated with metabolism pathways and that metabolism pathways were the richest pathways in KEGG enrichment analysis, which is consistent with our findings [27]. In contrast, Su study of genes and their associated signalling pathways before and after primordial follicle initiation in mice found that differential target genes involved in ovarian follicle development were more enriched in the MAPK signalling pathway [28].
Asma et al. found that granulosa cells were able to act as a barrier to protect oocytes from the extra-ovarian microenvironment through metabolic and signalling interactions [29].Lin et al. found significant changes in the transcriptional activity of genes belonging to the categories of neuroactive ligand-receptor interactions and metabolic pathways in mice with premature ovarian failure, suggesting that metabolic pathways may play an important role in premature ovarian failure [30]. A previous study reported that women with polycystic ovary syndrome had fertility changes, primarily related to metabolic disorders, such as insulin resistance, hyperinsulinemia, and/or dyslipidemia [31]. Such a correlation reveals that metabolism pathways are vital in regulating the reproductive system.
MAPK belongs to a big family of serine-threonine kinases, forming the major signalling pathways of cell proliferation from the cell surface to the nucleus. MAPK cascading is the critical signalling pathway that regulates several cell processes, including proliferation, differentiation, apoptosis and stress response [32]. It was found that when the MAPK signalling pathway was blocked, progesterone-induced MAPK activation was prevented, which in turn inhibited oocyte maturation [33]. Vossler et al. demonstrated that MAPK/ERK signalling pathways are related to follicle-stimulating hormone-induced granulosa cells' steroid biosynthesis [34].
Lysosome is vital to cell survival. It can eliminate injured, oxidized or dysfunctional organelles in bodies. Therefore, lysosome dysfunction can lead to oxidative stress [35]. A previous study pointed out that the lysosome metabolism pathway and signalling pathways of many lysosome proteases, such as Cathepsins and lysosome-associated membrane glycoprotein (Lamp), participate in cell apoptosis [35]. In studies using the cell apoptosis model, transparency of the lysosome membrane occurs in the early stage of apoptosis. It is the key cause of cell apoptosis and the death of apoptotic cells [36].