MicroRNA-132 promotes estradiol synthesis in ovarian granulosa cells via translational repression of Nurr1.
BACKGROUND
Estrogen synthesis is an important function of the mammalian ovary. Estrogen plays important roles in many biological processes, including follicular development, oocyte maturation and endometrial proliferation, and dysfunctions in estrogen synthesis contribute to the development of polycystic ovary syndrome and premature ovarian failure. Classical signaling cascades triggered by follicle-stimulating hormone induce estrogen synthesis via the upregulation of Cyp19a1 in granulosa cells (GCs). This study aimed to determine the effect of microRNA-132 (miR-132) on estradiol synthesis in GCs.


METHODS
Primary mouse GCs were collected from ovaries of 21-day-old immature ICR mice through follicle puncture. GCs were cultured and treated with the stable cyclic adenosine monophosphate analog 8-Br-cAMP or transfected with miR-132 mimics, Nurr1-specific small interfering RNA oligonucleotides and Flag-Nurr1 plasmids. Concentrations of estradiol 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 Cyp19a1, Cyp11a1 and an orphan nuclear receptor-Nurr1 expression in GCs. Direct suppression of Nurr1 via its 3'-untranslated region by miR-132 were further verified using luciferase reporter assays.


RESULTS
The expression level of miR-132 in cultured mouse GCs was significantly elevated during 48 h of treatment with 8-Br-cAMP. The synthesis of estradiol increased after the overexpression of miR-132 in mouse GCs. The real-time PCR results demonstrated that miR-132 induced the expression of Cyp19a1 significantly. Nurr1, an orphan nuclear receptor that suppresses Cyp19a1 expression, was found to be a direct target of miR-132. Nurr1 was suppressed by miR-132, as indicated by a luciferase assay and Western blotting. The knockdown of Nurr1 primarily elevated the synthesis of estradiol and partially attenuated the miR-132-induced estradiol elevation, and the ectopic expression of Flag-Nurr1 abrogated the stimulatory effect of miR-132 on estradiol synthesis in mouse GCs.


CONCLUSIONS
Our findings suggest that miR-132 is involved in the cAMP signaling pathway and promotes estradiol synthesis via the translational repression of Nurr1 in ovarian GCs.
Reproductive Medicine Center, Department of Obstetrics and Gynecology, Nanjing Drum Tower Hospital, Nanjing University Medical School, Nanjing, China. shgenwu@163.com.
Shaogen
S
Wu
Reproductive Medicine Center, Department of Obstetrics and Gynecology, Nanjing Drum Tower Hospital, Nanjing University Medical School, Nanjing, China. stevensunz@163.com.
Haixiang
H
Sun
Reproductive Medicine Center, Department of Obstetrics and Gynecology, Nanjing Drum Tower Hospital, Nanjing University Medical School, Nanjing, China. yuanxiao-0223@163.com.
Qun
Q
Zhang
Reproductive Medicine Center, Department of Obstetrics and Gynecology, Nanjing Drum Tower Hospital, Nanjing University Medical School, Nanjing, China. jiangyue85@163.com.
Yue
Y
Jiang
Reproductive Medicine Center, Department of Obstetrics and Gynecology, Nanjing Drum Tower Hospital, Nanjing University Medical School, Nanjing, China. fountain571@163.com.
Ting
T
Fang
New York-Presbyterian/Weill Cornell Medical Center, New York, NY, USA. ihcui87@gmail.com.
Isabelle
I
Cui
Reproductive Medicine Center, Department of Obstetrics and Gynecology, Nanjing Drum Tower Hospital, Nanjing University Medical School, Nanjing, China. yanguijun55@163.com.
Guijun
G
Yan
Reproductive Medicine Center, Department of Obstetrics and Gynecology, Nanjing Drum Tower Hospital, Nanjing University Medical School, Nanjing, China. glyyhuyli@163.com.
Yali
Y
Hu
D000818:Animals; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000494:pharmacology; D004958:Estradiol / Q000096:biosynthesis* / Q000235:genetics; D005260:Female; D006107:Granulosa Cells / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008813:Mice, Inbred ICR; D035683:MicroRNAs / Q000096:biosynthesis* / Q000235:genetics; D057126:Nuclear Receptor Subfamily 4, Group A, Member 2 / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics; D006080:Ovarian Follicle / Q000187:drug effects / Q000378:metabolism*
C546976:MIRN132 microRNA, mouse; D035683:MicroRNAs; C539883:Nr4a2 protein, mouse; D057126:Nuclear Receptor Subfamily 4, Group A, Member 2; C016276:8-bromocyclic GMP; D004958:Estradiol; D006152:Cyclic GMP