Effects of miR-101-3p on goat granulosa cells in vitro and ovarian development in vivo via STC1

Abstract

Background MiRNAs act as pivotal post-transcriptional gene mediators in the regulation of diverse biological processes, including proliferation, development and apoptosis. Our previous study has showed that miR-101-3p is differentially expressed in dairy goat ovaries compared single with multiple litters. The objective of this research was to explore the potential function and molecular mechanism of miR-101-3p via its target STC1 in goat ovarian growth and development. Results cDNA libraries were constructed using goat granulosa cells transfected with miR-101-3p mimics and negative control by RNA-sequencing. In total, 142 differentially expressed unigenes (DEGs) were detected between two libraries, including 78 down-regulated and 64 up-regulated genes. GO and KEGG enrichment analysis showed the potential impacts of DEGs on ovarian development. STC1 was singled out from DEGs for further research owing to it regulates reproductive-related processes. In vitro, bioinformatics analysis and 3′-UTR assays confirmed that STC1 was a target of miR-101-3p. ELISA was performed to detect the estrogen (E2) and progesterone (P4) levels. CCK8, EdU and flow cytometry assays were performed to detect the proliferation and apoptosis of granulosa cells. Results showed that miR-101-3p regulated STAR, CYP19A1, CYP11A1 and 3β-HSD steroid hormone synthesis-associated genes by STC1 depletion, thus promoted E2 and P4 secretions. MiR-101-3p also affected the key protein PI3K, PTEN, AKT and mTOR in PI3K-AKT pathway by STC1, thereby suppressing proliferation and promoting apoptosis of granulosa cells. In vivo, the distribution and expression levels of miR-101-3p in mouse ovaries were determined through fluorescence in situ hybridisation (FISH). Immunohistochemistry results showed that STC1 expression was suppressed in mouse ovaries in miR-101-3p-agonist and siRNA-STC1 groups. Small and stunted ovarian fragments, decreased numbers of follicles at diverse stages were observed using Hematoxylin-eosin (HE) staining, thereby showing unusual ovarian development after miR-101-3p overexpression or STC1 depletion. Inhibition of miR-101-3p manifested opposite results. Conclusions Taken together, our results demonstrated a regulatory mechanism of miR-101-3p via STC1 in goat granulosa cells, and offered the first in vivo example of miR-101-3p and STC1 functions required for ovarian development.

Integrated Analysis of mRNA- and miRNA-Seq in the Ovary of Rare Minnow Gobiocypris rarus in Response to 17α-Methyltestosterone

“…The miR-183 cluster (miR-96/183)-foxo3 pair presented in this study revealed that its pathway may result in damage to the central nervous system (Li et al, 2010), immune impairment, or even cancer (Dambal et al, 2015;Ichiyama and Dong, 2019;Zou et al, 2019). The latest study showed that miR-101 regulated STAR, CYP19A1, CYP11A1, and 3β-HSD steroid hormone synthesis-associated genes by STC1 depletion, thus, promoting E2 secretions (An et al, 2020). These results suggest that MT regulates related gene expression by interfering with miRNAs, thereby, injuring the ovary.…”

TLR7/8 signalling affects X-sperm motility via the GSK3 α/β-hexokinase pathway for the efficient production of sexed dairy goat embryos

“…The phosphoinositide 3-kinases (PI3Ks), a family of lipid kinases, participate in multiple signalling pathways in cells, including cell proliferation, migration, survival, metabolism, and intracellular trafficking [ 38 ]. AKT is a key downstream stimulant of PI3K and is closely related to sperm motility [ 39 , 40 ]. Li et al (2020) focused on the relationship between sperm motility and PI3K/AKT activity in mice and found that sperm motility decreased notably after blocking AKT activity [ 41 ].…”

Comparison of MicroRNA Profiles in Extracellular Vesicles from Small and Large Goat Follicular Fluid

“…The PI3K-Akt pathway is regulated by several miRNAs, such as miR-202-5p [28], miR-101 [29,30], and miR-22-3p [31], which are upregulated in LFF-EVs. It was reported that exosomes promote GCs' proliferation through the P13K-AKT signaling [12] and MAPK signaling pathways [24].…”

“…These signaling pathways play key roles in the proliferation and apoptosis of mammalian cells during follicular growth and in the promotion of primordial follicle activation [ 26 , 27 ]. The PI3K-Akt pathway is regulated by several miRNAs, such as miR-202-5p [ 28 ], miR-101 [ 29 , 30 ], and miR-22-3p [ 31 ], which are upregulated in LFF-EVs. It was reported that exosomes promote GCs’ proliferation through the P13K-AKT signaling [ 12 ] and MAPK signaling pathways [ 24 ].…”