In mammals, microRNAs (miRNAs) play key roles in multiple biological processes by regulating the expression of target genes.  Studies have found that the levels of miR-370-5p expression differ significantly in the skins of sheep with different hair colors; however, its function remains unclear.  In this study, we investigated the roles of miR-370-5p in sheep melanocytes and found that the overexpression of miR-370-5p significantly inhibited cell proliferation (P<0.01), tyrosinase activity (P=0.001) and significantly reduced (P<0.001) melanin production.  Functional prediction revealed that the 3´-untranslated region (UTR) of MAP3K8 has a putative miR-370-5p binding site, and the interaction between these two molecules was confirmed using luciferase reporter assays.  In situ hybridization assays revealed that MAP3K8 is expressed in the cytoplasm of melanocytes.  The results of quantitative RT-PCR and Western blotting analyses revealed that overexpression of miR-370-5p in melanocytes significantly inhibits (P<0.01) MAP3K8 expression via direct targeting of its 3´ UTR.  Inhibition of MAP3K8 expression by siRNA-MAP3K8 transfection induced a significant inhibition (P<0.01) of melanocyte proliferation and significant reduction (P<0.001) in melanin production, which is consistent with our observations for miR-370-5p.  Target gene rescue experiments indicated that the expression of MAP3K8 in melanocytes co-transfected with miR-370-5p and MAP3K8-cDNA (containing sites for the targeted binding to miR-370-5p) was significantly rescued (P≤0.001), which subsequently promoted significant increases in cell proliferation (P<0.001) and melanin production (P<0.01).  Collectively, these findings indicate that miR-370-5p plays a functional role in inhibiting sheep melanocyte proliferation and melanogenesis by downregulating the expression of MAP3K8.  

MicroRNA (miRNA) has vital regulatory effects on the proliferation, differentiation and secretion of ovarian granulosa cells, but the role of miR-99a-5p in goat ovarian granulosa cells (GCs) is unclear.  Both miR-99a-5p and Frizzled-5 (FZD5) were found to be expressed in GCs in goat ovaries via fluorescence in situ hybridization and immunohistochemistry, respectively, and FZD5 was verified (P<0.001) as a target gene of miR-99a-5p by double luciferase reporter gene experiments.  Furthermore, FZD5 mRNA and protein expression were both found to be regulated (P<0.05) by miR-99a-5p in GCs.  Moreover, the overexpression of miR-99a-5p or knockdown of FZD5 suppressed (P<0.05) estradiol and progesterone secretion from the GCs, as determined by ELISA.  In summary, miR-99a-5p inhibits target gene FZD5 expression and estradiol and progesterone synthesis in GCs.  Our study thus provides seminal data and new insights into the regulatory mechanisms of follicular development in the goat and other animals.

N6-methyladenosine (m6A) plays a key role in mammalian early embryonic development and cell lineage differentiation. However, the role and mechanisms of 18S ribosomal RNA (rRNA) m6A methyltransferase METTL5 in early embryonic development remain unclear. Here, we found that 18S rRNA m6A methyltransferase METTL5 plays an important role in porcine early embryonic development. METTL5 knockdown and overexpression significantly reduced the developmental efficiency of porcine early embryos and impaired cell lineage allocation. METTL5 knockdown apparently decreased the global translation efficiency in blastocyst, while METTL5 overexpression increased the global translation efficiency. Furthermore, METTL5 knockdown did not affect the abundance of CDX2 mRNA, but resulted in a significant reduction in CDX2 protein levels. Moreover, the low developmental efficiency and abnormal lineage distribution of METTL5 knockdown embryos could be rescued by CDX2 overexpression. Collectively, our results demonstrated that 18S rRNA methyltransferase METTL5 regulates porcine early embryonic development via modulating the translation of CDX2.

Skin and hair pigmentation in animals involve intricate regulatory processes. Circular RNA-microRNA (circRNA-miRNA) networks play vital roles in various biological processes, although their involvement in pigmentation has been underexplored. This study focused on circKIF27 expression, which differs significantly in melanocytes isolated from white and brown Boer coat-colored skin, yet its function remains unclear. Here, we investigated the roles of circKIF27 in melanocytes. In situ hybridization assays demonstrated that circKIF27 is expressed in the cytoplasm of melanocytes. qRT-PCR results revealed differential expression levels of circKIF27 in various tissues of male and female goats. Functional analysis showed that circKIF27 overexpression in melanocytes significantly reduces melanin production (P<0.01) and inhibits cell proliferation (P<0.0001). Bioinformatics analysis identified a putative miR-129-5p binding site on circKIF27, and luciferase reporter assays confirmed their interaction. Overexpression of miR-129-5p in melanocytes enhances melanin production (P<0.01) and promotes cell proliferation (P<0.05). Further analysis revealed that TGIF2 possesses two potential miR-129-5p binding sites, and miR-129-5p overexpression in melanocytes significantly inhibits TGIF2 expression (P<0.0001), suggesting a targeted regulatory relationship between these two molecules. Silencing TGIF2 expression via siRNA-TGIF2 transfection leads to increased melanocyte proliferation (P<0.0001) and increased melanin production (P<0.01). These findings highlight the involvement of the circRNA-miRNA network in pigmentation, offering new insights into the molecular mechanisms underlying pigmentation and guiding animal hair color breeding strategies.