In our previous study, we compared the miRNA expression profile of the growing and fully grown porcine oocytes based on the follicle size using small RNA high-throughput sequencing technology [16]. Results showed that miR-152 was among the significantly upregulated miRNAs in the large fully grown oocytes compared to the small growing ones. To verify the role of miR-152 in determining the developmental competence of porcine oocytes, in this study we managed to manipulate the expression level of miR-152 in porcine COCs during the maturation process. We mainly found that a suppressed expression of miR-152 during oocyte maturation significantly improved the blastocyst rate. Recently, it has been reported that the miRNAs expression pattern is highly correlated with oocyte quality and developmental competence [25]. Moreover, stage-specific and dynamic changes in the expression pattern of several miRNAs were observed during oocyte [26–28] and embryo development [29, 30], indicating a regulatory role of miRNAs in oogenesis and embryogenesis. Functional analysis studies revealed the specific function of several miRNAs as post-transcriptional regulators during oocyte and embryo development. For instance in pigs, miR-224 has been reported to be a regulator of the CCs function and subsequently affect oocyte maturation [31]. Moreover, manipulating the expression level of miR-155 in mice COCs proved its role in CCs expansion, oocyte maturation, and cleavage rate [32]. Several other miRNAs, such as let-7c, miR-27a, and miR-322, have been investigated and showed a complex regulatory role of oocyte developmental competence through functional regulation of the surrounding follicular cells [33]. These evidences highlighted the substantial roles of miRNAs in mammalian reproduction throughout folliculogenesis, oogenesis, and embryo development (for a review, see [4]).
It is well known that miR-152 plays an important role as a tumor suppressor in human cancer [34]. Several mechanisms have been demonstrated to explain this role, including the epigenetic regulation of miR-152/DNMT1 [35], the inhibition of cell proliferation [36], and suppression of the PI3K/Akt and MAPK signaling pathways [37]. In the current study, we successfully manipulated the expression level of miR-152 in porcine COCs via the transfection of miR-152 mimics and inhibitors. The quantitative analysis of PCR data showed a significant reduction and increase in the miR-152 expression level in inhibitor and mimic oocyte groups, respectively. Target gene prediction and pathway analysis revealed that miR-152 could be a potential candidate for controlling oocyte development through targeting different essential pathways and genes, including IGF1 and its receptor IGF1R. Gene expression analysis of oocyte groups showed that IGF1 was significantly upregulated in the inhibitor group and downregulated in the mimic group, while IGF1R was significantly upregulated in the inhibitor group compared to the control one. This may indicate a potential regulatory mechanism between miR-152 and the IGF system during oocyte development. Several studies reported that IGF-1 is involved in the regulation of follicular, oocyte, and embryonic development and the proliferation of follicular cells [38–40]. A significant correlation between IGF1 expression and blastocyst formation has been observed in mice [41]. In other mammalian species, including pigs, it has been confirmed that IGF-1 improves in vitro maturation, fertilization, and blastocyst rates [42–46]. In support of our results, the association between IGF-1 and miR-152 expression has been previously demonstrated in human oocytes, since the culturing of immature oocytes in the presence of IGF-1 significantly reduced the expression of miR-152 [47]. Moreover, in cancer research, it was suggested that miR-152 is involved in the IGF-1-mediated miR-152/PKM2/β-catenin regulatory circuit that regulates cell proliferation and angiogenesis [48]. Additionally, the overexpression of miR-152 inhibits breast cancer cell proliferation via targeting IGF1R and IRS1 and suppressing their downstream AKT and MAPK/ERK signaling pathways [37]. We also measured the expression level of two IGF-binding proteins (IGFBP6 and IGFBP7) in the treated oocytes. The results showed a significant reduction in IGFBP6 in the inhibition group compared to other groups. IGF-binding proteins control the bioavailability of IGFs to their corresponding cells [49]. The expression levels of IGFBP-2 mRNA and protein were reduced during oocyte maturation in bovines [50]. Previously we reported a significant decrease in IGFBP2, 6, and 7 in the high- compared to low-competence porcine oocytes [51]. These results in addition to our current findings may indicate that the improved blastocyst rate after the inhibition of miR-152 in the oocyte is due to the higher IGF bioavailability through the upregulation of IGF1 and IGF1R and the reduction in IGFBP6.
Pathway analysis identified several signaling pathways (including PI3K/AKT, TGFβ, Hippo, FoxO, and Wnt signaling) that are enriched in predicted miR-152 target genes. The expression of these signaling pathway components is essential for the oocyte to embryo transition and during the early implantation stage, including blastocyst formation [52, 53]. For instance, the inhibition of PI3K or AKT, components of the PI3K/AKT signaling pathway, eventually produces a significant decrease in embryo development and blastocyst rate in bovines [54, 55]. In this study, blastocysts developed from the mimic-treated oocytes exhibited an increase in miR-152 expression compared to the other groups. This group represents the lowest rate of blastocyst formation, which indicates a negative effect of miR-152 on embryonic development. In agreement with our results, Nie et al. [56] reported that the transfection of specific miR-152 mimics into the endometrial epithelial cells led to impaired embryonic development and implantation. The same blastocyst group exhibited a reduction in the SOD1 expression pattern, suggesting a lower antioxidant capacity of these blastocysts. Recently, it has been reported that miR-152 expression is positively correlated with oxidative stress in HL-2 cells, in which a dose-dependent high expression of miR-152 was observed after treatment with hydrogen peroxide (H2O2) [57]. Although the oxidative stress and the reduction in SOD levels could lead to cell apoptosis in preimplantation embryos [58], the expression of the antiapoptotic gene BCL2 was significantly upregulated in the blastocysts of the mimic group compared to the inhibitor and negative control groups. In agreement with our results, Cao et al. reported that the transfection of miR-152 mimics significantly increases BCL2 expression and inhibits apoptosis induced by hypoxia in HBMECs cells [59].