Study On MiR-26a/b-mediated Molecular Mechanisms On Progesterone Receptor Regulated By Estrogen In Granulosa Cells

In female mammals,ovulation is a very important physiological activities which might be controlled by many coordinated factors.Because of the extensive range and cascade reaction,the hormonal become one of the major regulatory factors in ovulation.Normal ovulation not only regulated by follicle stimulating hormone and luteinizing hormone,but also by estrogen and progesterone.In mammals,estrogen and progesterone play physiological roles rely on their specific receptors.Knockout mice have confirmed the normal expression of progesterone receptor is significant for various reproductive function include ovulation.In recent years,through RNA-Seq and CHIP-Seq,a large number of progesterone receptor downstream genes has been selected,among them,the peroxidase hyperplasia activated receptor ? and matrix metalloproteinases 1 were considered to be key signal molecules in ovulation.In addition,during the process of ovulation,the concentration of estrogen and progesterone in blood rendering certain relevance,whether the regulation of each other is not yet clear.At the same time,estrogen can influence the expression of progesterone receptor in the cell has been confirmed,but the molecular mechanism remains to be studied.There is a lot of research has shown that estrogen can cause the change of the miRNA expression and some miRNAs can also regulate progesterone receptor.In addition,during the follicular development,miR-26 a/b in granulosa cells maintain high levels of expression and there are differences in different periods.This study intends to explore in the granulosa cells during ovulation whether miR-26 a/b could mediate estrogen regulation of progesterone receptor.The results show that the primary granular cells of mice,after 24 h estrogen treatment,the pri-miR-26 a/b,the pre-miR-26 a/b and miR-26 a/b expression were significantly lower(p?0.05).In addition,progesterone receptor and its downstream genes expression of are markedly raised at mRNA and protein levels.Through bioinformatics analysis,a targeted sequence of miR-26 a/b in progesterone receptor 3 '-UTR was found.The double fluorescent report experiments demonstrated that mi R-26 a/b could regulate the progesterone receptor at post-transcriptional level.In order to confirm miR-26 a/b can mediate estrogen regulation of progesterone receptor in granular cells,this study selected the human granular cell line(KGN)as the experimental object.After 24 h estrogen treatment,the pri-miR-26 a/b,the pre-miR-26 a/b and miR-26 a/b were significantly lower,furthermore,progesterone receptor and its downstream genes expression of are significantly higher.When estrogen signal pathway was blocked by tamoxifen or shRNA,estrogen could not cause dysregulation of miRNA and progesterone receptor.Furthermore,when transfect mi R-26 a/b minics or inhibitors,the expression of progesterone receptor would be changed.In order to simulate the mature process of miRNA more accurately,miR-26 a/b expression plasmid were constructed.Transient transfection of miRNA expression vector can inhibit estrogen regulation of progesterone receptor.In conclusion,the results showed that in the granular cells of female mammals,the progesterone receptor,response of estrogen signaling molecules may be mediated by miR-26 a/b.It suggested that regulation of progesterone receptor in ovulation does not only rely on progesterone.This study provides a new view for exploring the hormone regulation in ovulation process...