Guijun Yan

MicroRNA-145 Protects Cardiomyocytes against Hydrogen Peroxide (H2O2)-Induced Apoptosis through Targeting the Mitochondria Apoptotic Pathway

MicroRNAs, a class of small and non-encoding RNAs that transcriptionally or post-transcriptionally modulate the expression of their target genes, has been implicated as critical regulatory molecules in many cardiovascular diseases, including ischemia/reperfusion induced cardiac injury. Here, we report microRNA-145, a tumor suppressor miRNA, can protect cardiomyocytes from hydrogen peroxide (H2O2)-induced apoptosis through targeting the mitochondrial pathway. Quantitative real-time PCR (qPCR) demonstrated that the expression of miR-145 in either ischemia/reperfused mice myocardial tissues or H2O2-treated neonatal rat ventricle myocytes (NRVMs) was markedly down-regulated. Over-expression of miR-145 significantly inhibited the H2O2-induced cellular apoptosis, ROS production, mitochondrial structure disruption as well as the activation of key signaling proteins in mitochondrial apoptotic pathway. These protective effects of miR-145 were abrogated by over-expression of Bnip3, an initiation factor of the mitochondrial apoptotic pathway in cardiomyocytes. Finally, we utilized both luciferase reporter assay and western blot analysis to identify Bnip3 as a direct target of miR-145. Our results suggest miR-145 plays an important role in regulating mitochondrial apoptotic pathway in heart challenged with oxidative stress. MiR-145 may represent a potential therapeutic target for treatment of oxidative stress-associated cardiovascular diseases, such as myocardial ischemia/reperfusion injury.

MicroRNA-181a Suppresses Mouse Granulosa Cell Proliferation by Targeting Activin Receptor IIA

Activin, a member of the transforming growth factor-β superfamily, promotes the growth of preantral follicles and the proliferation of granulosa cells. However, little is known about the role of microRNAs in activin-mediated granulosa cell proliferation. Here, we reported a dose- and time-dependent suppression of microRNA-181a (miR-181a) expression by activin A in mouse granulosa cells (mGC). Overexpression of miR-181a in mGC suppressed activin receptor IIA (acvr2a) expression by binding to its 3′-untranslated region (3′-UTR), resulting in down-regulation of cyclin D2 and proliferating cell nuclear antigen expression, leading to inhibition of the cellular proliferation, while overexpression of acvr2a attenuated the suppressive effect of miR-181a on mGC proliferation. Consistent with the inhibition of acvr2a expression, miR-181a prevented the phosphorylation of the activin intracellular signal transducer, mothers against decapentaplegic homolog 2 (Smad2), leading to the inactivation of activin signaling pathway. Interestingly, we found that miR-181a expression decreased in ovaries of mice at age of 8, 12, and 21 days, as compared with that in ovaries of 3-day old mice, and its level was reduced in preantral and antral follicles of mice compared with that in primary ones. Moreover, the level of miR-181a in the blood of patients with premature ovarian failure was significantly increased compared with that in normal females. This study identifies an interplay between miR-181a and acvr2a, and reveals an important role of miR-181a in regulating granulosa cell proliferation and ovarian follicle development.

MicroRNA-133b stimulates ovarian estradiol synthesis by targeting Foxl2.

Forkhead L2 (Foxl2) is expressed in ovarian granulosa cells and participates in steroidogenesis by transcriptionally regulating target genes such as steroidogenic acute regulatory protein (StAR) and CYP19A1. In this study, a direct link between microRNA‐133b (miR‐133b) and Foxl2‐mediated estradiol release in granulosa cells was established. miR‐133b was involved in follicle‐stimulating hormone (FSH)‐induced estrogen production. Luciferase assays confirmed that miR‐133b was bound to the 3′ untranslated region (3′UTR) of Foxl2 mRNA. Consistent with this finding, miR‐133b overexpression reduced the Foxl2 levels. Furthermore, miR‐133b inhibited Foxl2 binding to the StAR and CYP19A1 promoter sequences. These results demonstrate that miR‐133b down‐regulates Foxl2 expression in granulosa cells by directly targeting the 3′UTR, thus inhibiting the Foxl2‐mediated transcriptional repression of StAR and CYP19A1to promote estradiol production.

MicroRNA-145 suppresses mouse granulosa cell proliferation by targeting activin receptor IB

MicroRNAs (miRNAs) are a class of 21‐ to 25‐nucleotide non‐coding RNAs, some of which are important gene regulators involved in folliculogenesis. In this study, we used CCK‐8, real‐time PCR and Western blot assays to demonstrate that miR‐145 inhibits mouse granulosa cell (mGC) proliferation. Combined with the results of luciferase reporter assays that studied the 3′‐untranslated region of ACVRIB mRNA, these assays identified ACVRIB as a direct target of miR‐145. The ectopic expression of miR‐145 reduced the levels of both ACVRIB mRNA and protein and also interfered with activin‐induced Smad2 phosphorylation. Altogether, this study revealed that miR‐145 suppresses mGC proliferation by targeting ACVRIB.

MicroRNA miR-302 inhibits the tumorigenicity of endometrial cancer cells by suppression of Cyclin D1 and CDK1.

MicroRNA miR-302 has been found to induce some tumor cell lines to transdifferentiate into miRNA-induced pluripotent stem cells (mirPS), thereby inhibiting tumor cell proliferation and reducing tumorigenicity. This study firstly found that miR-302 inhibited the proliferation and migration of endometrial cell line, Ishikawa and HEC-1-B, and arrested cell cycle at the G2/M phase. In addition, miR-302 inhibited tumorigenicity in immunodeficient mice transplanted with Ishikawa cells. Microarray and Western blotting results showed that miR-302 significantly inhibited CDK1 and Cyclin D1 gene expression in Ishikawa cells. MiR-302 directly targeted Cyclin D1, but indirectly regulated CDK1 gene expression.

Single Administration of Ultra-Low-Dose Lipopolysaccharide in Rat Early Pregnancy Induces TLR4 Activation in the Placenta Contributing to Preeclampsia

Balanced immune responses are essential for the maintenance of successful pregnancy. Aberrant responses of immune system during pregnancy increase the risk of preeclampsia. Toll-like receptor 4 (TLR4) plays a crucial role in the activation of immune system at the maternal-fetal interface. This study aimed to generate a rat model of preeclampsia by lipopolysaccharide (LPS, a TLR4 agonist) administration on gestational day (GD) 5 as rats are subjected to placentation immediately after implantation between GDs 4 and 5, and to assess the contribution of TLR4 signaling to the development of preeclampsia. Single administration of 0.5 μg/kg LPS significantly increased blood pressure of pregnant rats since GD 6 (systolic blood pressure, 124.89 ± 1.79 mmHg versus 119.02 ± 1.80 mmHg, P < 0.05) and urinary protein level since GD 9 (2.02 ± 0.29 mg versus 1.11 ± 0.18 mg, P < 0.01), but barely affected blood pressure or proteinuria of virgin rats compared with those of saline-treated pregnant rats. This was accompanied with adverse pregnancy outcomes including fetal growth restriction. The expression of TLR4 and NF-κB p65 were both increased in the placenta but not the kidney from LPS-treated pregnant rats, with deficient trophoblast invasion and spiral artery remodeling. Furthermore, the levels of inflammatory cytokines were elevated systemically and locally in the placenta from pregnant rats treated with LPS. TLR4 signaling in the placenta was activated, to which that in the placenta of humans with preeclampsia changed similarly. In conclusion, LPS administration to pregnant rats in early pregnancy could elicit TLR4-mediated immune response at the maternal-fetal interface contributing to poor early placentation that may culminate in the preeclampsia-like syndrome.

PCAF Impairs Endometrial Receptivity and Embryo Implantation by Down-Regulating β3-Integrin Expression via HOXA10 Acetylation

BACKGROUNDnHomeobox A10 (HOXA10), a key transcription factor, plays a critical role in endometrial receptivity by regulating the expression of downstream target genes, such as β3-integrin (ITGB3), but little is understood about the mechanisms of the posttranslational modification of HOXA10 during embryo implantation.nnnOBJECTIVEnThe aim of this study was to assess the effect of HOXA10 acetylation by p300/CREB-binding protein-associated factor (PCAF) in the embryo implantation process.nnnMETHODSnThe association of HOXA10 with PCAF was detected by coimmunoprecipitation, Western blotting, and confocal immunofluorescent assays. A luciferase reporter assay, Western blotting, quantitative real-time PCR, and chromatin immunoprecipitation techniques were used to determine the effect of PCAF on HOXA10 protein stability and the HOXA10-mediated regulation of ITGB3 expression. HOXA10-PCAF association on embryo implantation was evaluated using a BeWo spheroid attachment assay. PCAF expression in the eutopic endometrium of women with endometriosis and fertile controls was measured by Western blotting technique.nnnRESULTSnPCAF was identified as an HOXA10-interacting protein and inhibited HOXA10-mediated ITGB3 transcription via acetylating HOXA10 at K338 and K339. Overexpressing or knocking down PCAF in Ishikawa cells showed that PCAF not only down-regulated HOXA10-mediated ITGB3 protein expression but also diminished HOXA10-mediated embryo adhesiveness by acetylating HOXA10 (P < .05). Furthermore, we found aberrantly high PCAF expression in the eutopic endometrium of women with a diagnosis of endometriosis compared with the fertile controls (P < .05).nnnCONCLUSIONSnThese observations demonstrate that 1) HOXA10 associates with and is acetylated by PCAF at lysines K338 and K339 in Ishikawa cells and 2) HOXA10-PCAF association impairs embryo implantation by inhibiting ITGB3 protein expression in endometrial epithelial cells.

FSH acts on the proliferation of type A spermatogonia via Nur77 that increases GDNF expression in the Sertoli cells.

The molecular mechanism responsible for the regulation of GDNF in Sertoli cells remains largely unknown. In the present study, FSH induced the expression of Nur77 and GDNF in mouse testis tissue and human fetal Sertoli cells. Moreover, FSH increased the number of A spermatogonia co‐cultured with Sertoli cells. In the additional assays, Nur77 was observed to directly regulate GDNF transcription. Furthermore, overexpression of Nur77 and siRNA‐mediated knockdown of Nur77 affected levels of GDNF mRNA and protein in primary human fetal Sertoli cells. These results indicate that FSH‐induced Nur77 regulates the expression of GDNF in Sertoli cells to stimulate the proliferation of A spermatogonia in vitro.

The orphan nuclear receptor Nur77 regulates decidual prolactin expression in human endometrial stromal cells

Prolactin (PRL) is synthesized and released by several extrapituitary tissues, including decidualized stromal cells. Despite the important role of decidual PRL during pregnancy, little is understood about the factors involved in the proper regulation of decidual PRL expression. Here we present evidence that the transcription factor Nur77 plays an active role in decidual prolactin expression in human endometrial stromal cells (hESCs). Nur77 mRNA expression in hESCs was significantly increased after decidualization stimulated by 8-Br-cAMP and medroxyprogesterone acetate (MPA). Adenovirus-mediated overexpression of Nur77 in hESCs markedly increased PRL mRNA expression and enhanced decidual PRL promoter (dPRL/-332Luc) activity in a concentration-dependent manner. Furthermore, knockdown of Nur77 in hESCs significantly decreased decidual PRL promoter activation and substantially attenuated PRL mRNA expression and PRL secretion (P < 0.01) induced by 8-Br-cAMP and MPA. These results demonstrate that Nur77 is a novel transcription factor that contributes significantly to the regulation of prolactin gene expression in human endometrial stromal cells.

MicroRNA-132 promotes estradiol synthesis in ovarian granulosa cells via translational repression of Nurr1

BackgroundEstrogen 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.MethodsPrimary 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.ResultsThe expression level of miR-132 in cultured mouse GCs was significantly elevated during 48xa0h 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.ConclusionsOur 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.