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.