Study On The Functions Of MicroRNA-132 In The Ovarian Granulosa Cells

Chapter One:MicroRNA-132 promotes estradiol synthesis of ovarian granulosa cells by translational repression of NurrlObjective:To study the transcriptional induction of microRNA-132 (miR-132) by follicle-stimulating hormone (FSH) pathway in ovarian granulosa cells, and the effect on estrogen synthesis of granulosa cells stimulated by miR-132 as well as the molecular mechanism underlying.Materials and Methods:Primary mouse granulosa cells (mGCs) were collected from ovaries of 21-day-old immature ICR mice through follicle puncture. mGCs were cultured and treated with 8-Br-cAMP or transfected with miR-132 mimics, Nurrl-specific siRNA oligonucleotides and Flag-Nurrl plasmids. Concentrations of estradiol (E2) and progesterone in culture medium were determined by an automated chemiluminescence-based assay. Quantitative real time PCR and western blot were performed to identify the effect of miR-132 on Cypl9al, Cypllal and an orphan nuclear receptor-Nurrl expression in mGCs. The 3’-UTR (3’-untranslated region) harboring putative binding site of miR-132 was cloned downstream of luciferase gene, and luciferase reporter assay was utilized to detemine the post-transcriptional suppression of Nurrl gene by miR-132.Results:The expression level of miR-132 in cultured mGCs was continuously induced by 8-Br-cAMP and reached the peak at 12 h (-5-fold increased, PO.05). The release of E2 promoted significantly more than 70%(P<0.01) after over-expressing of miR-132. However, no significant change of progesterone was detected. Real time PCR exhibited that miR-132 induced the expression of Cypl9al (2.8-fold increased, P<0.01) but not Cyp11a1, a key gene for progesterone synthesis. The repression of Nurrl protein level by miR-132 was identified by western blot without significant transcriptional alternation. miR-132 significantly repressed pmirGLO-luc-Nurrl 3’-UTR-WT luciferase activity (P<0.01), but not pmirGLO-luc-Nurrl 3’-UTR-MU with mutant binding sequences. Taken together, miR-132 translational suppresses Nurr1, which attenuates the inhibitory effect of Nurrl on Cypl9al expression through its PII promoter. Knock-down of endogenous Nurrl by siNurrl partly attenuated the effect of miR-132 and ectopic expression of Flag-Nurrl significantly abrogated the stimulation effect of miR-132 on E2 release.Conclusions:E2 biosynthesis of mGCs is promoted by miR-132 by upregulation of Cyp19a1. The induction of Cypl9al is indirectly regulated by miR-132 through suppressing the expression of Nurrl via directly targeting its 3’-UTR.Chapter Two:Apoptosis of ovarian granulosa cells is activated by microRNA-132 through targeting FoxalObjective:To study the pro-apoptosis effect of miR-132 through directly targeting Foxal and subsequently affects estrogen receptor (ER) pathway in mGC.Materials and Methods:mGCs were cultured and transfected with miR-132 mimics or negative control followed with detection of apoptosis of mGCs by Annexin V flow cytometry and Cell Death Detection ELISA. Protein levels of ERa and ERβ were determined using Western blot. The expression and distribution of Foxal in ovaries of 21 d postpartum mouse after superovulation by PMSG (5 IU i.h.) were observed by immunohistochemistry. Both luciferase reporter assay and Western blot were utilized to determined Foxal as the direct target of miR-132. Down-regulation endogenous Foxal using siFoxal further studied the apoptosis effect in mGCs. The nucleic localization of ERa in mGCs after transfected with miR-132 mimics was visulized using immunofluorescence.Results:After transfected with miR-132 mimics or negative control, apoptosis of mGCs was significantly induced in a dose-dependent manner (P<0.05) by miR-132. The expression of ERa in mGCs was down-regulated but not ERβ. The expression of Foxal in granulosa cells of activated follicles after superovulation was lower than that of normal controls. Luciferase reporter assay showed a 45% decrease (P<0.05) of luciferase activity of pmirGLO-luc-Foxal 3’-UTR by over expression of miR-132. Western blot confirmed the down-regulation of protein level of Foxal by miR-132. After silencing endogenous Foxal using siFoxal, the level of ERa was also down-regulated accompanied with induced apoptosis of mGCs (P<0.001). Over-expression of miR-132 in mGCs also enhanced the distribution of ERa in cytoplasm.Conclusions:miR-132 down-regulates the expression of ERa through directly suppressing Foxal, which leads to the apoptosis of mGCs.