MEL regulates miR-21 and let-7b through the STAT3 cascade in the follicular granulosa cells of Tibetan sheep

Abstract

Under physiological and pathological conditions, melatonin (MEL) can regulate microRNA (miRNA) expression. However, the mechanisms underlying the regulatory effects of MEL on miRNAs in ovaries are not understood. Firstly, by using fluorescence in situ hybridisation, we found that in ovaries and follicular granulosa cells (FGCs), MT1 co-located with miR-21 and let-7b. Additionally, immunofluorescence revealed that MT1, STAT3, c-MYC and LIN28 proteins co-located. The mRNA and protein levels of STAT3, c-MYC and LIN28 increased under treatment with 10−7 M MEL. MEL induced an increase in miR-21 and a decrease in let-7b.The LIN28/let-7b and STAT3/miR-21 axes are related to cell differentiation, apoptosis and proliferation. We explored whether the STAT3/c-MYC/LIN28 pathway was involved in miRNA regulation by MEL to explore the putative mechanism of the above relationship. AG490, an inhibitor of the STAT3 pathway, was added before MEL treatment. AG490 inhibited the MEL-induced increases in STAT3, c-MYC, LIN28 and MT1 and changes in miRNA. Through live-cell detection, we discovered that MEL enhanced the proliferation of FGCs. However, the ki67 protein levels decreased when AG490 was added in advance. Furthermore, the dual-luciferase reporter assay verified that STAT3, LIN28 and MT1 were target genes of let-7b. Furthermore, STAT3 and SMAD7 were target genes of miR-21. In addition, the protein levels of the STAT3, c-MYC, LIN28 and MEL receptors decreased when let-7b was overexpressed in FGCs. Overall, MEL might regulate miRNA expression through the STAT3 pathway. In addition, a negative feedback loop between the STAT3 and miR-21 formed; MEL and let-7b antagonized each other in FGCs. These findings may provide a theoretical basis for improving the reproductive performance of Tibetan sheep through MEL and miRNAs.