Haixiang Sun

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.

Regeneration of uterine horns in rats by collagen scaffolds loaded with collagen-binding human basic fibroblast growth factor.

Severe damages of uterine endometrium which prevent embryos from implantation and placentation finally often result in infertility or pregnant complications. There is lack of effective treatments due to the limitation of native materials available and complexity of the function and internal environment of uterus. In the present study, a collagen targeting basic fibroblast growth factor (bFGF) delivery system was constructed by a collagen membrane loaded with bFGF fused a collagen-binding domain (CBD) to the N-terminal which limits the diffusion of bFGF from collagen. We tested the bFGF delivery system in rats under the severe uterine damage model (partial rat uterine horn excision/reconstruction), and found this delivery system improved regeneration abilities of uterine endometrium and muscular cells, improved vascularization, as well as better pregnancy outcomes in rats. Therefore, this targeting delivery system may be an effective strategy for uterine tissue regeneration.

MicroRNA-155 is involved in the remodelling of human-trophoblast-derived HTR-8/SVneo cells induced by lipopolysaccharides

BACKGROUND A low dose injection of lipopolysaccharides (LPS) may induce pre-eclampsia-like symptoms in rats, and microRNA-155 (miR-155) is elevated in the placentas of patients with pre-eclampsia. Our goal was to investigate the association of miR-155 with pre-eclampsia and the pathways involved using human-trophoblast-derived cell line (HTR-8/SVneo) stimulated with LPS. METHODS We measured miR-155 in HTR-8/SVneo cells treated with LPS (25-800 ng/ml) using real-time PCR. Western blotting was used to study transcription factor activated protein 1 (AP-1) (JunB and FosB subunits) and nuclear factor (NF)-κB p65 in the HTR-8/SVneo cells and placentas from patients with pre-eclampsia. DNA precipitation assays and luciferase reporter analysis were used to evaluate the regulation of miR-155 by AP-1 and NF-κB. Cell migration was determined by scratch assay. Syncytialization of HTR-8/SVneo cells was analysed following transfection with miR-155. RESULTS miR-155 was increased together with AP-1 and NF-κB in HTR-8/SVneo cells incubated with low dose of LPS (≤100 ng/ml; P < 0.05 versus baseline). Both JunB/FosB and p65 were increased in placenta from women with severe pre-eclampsia versus a normal pregnancy, with elevated expression of miR-155 (P < 0.05). For specific DNA-binding sites upstream of BIC/miR-155 gene promoter, the AP-1 site was more important than the NF-κB site for increasing miR-155 in HTR-8/SVneo cells. The cells with enforced expression of miR-155 showed a reduced ability to migrate (P < 0.05) and an increased number of syncytiotrophoblast-like multinuclear cells (P < 0.05). CONCLUSIONS LPS may induce remodelling of the human-trophoblast-derived HTR-8/SVneo cells by increasing miR-155, acting in part through the AP-1 and NF-κB pathways.

Transplantation of bone marrow mesenchymal stem cells on collagen scaffolds for the functional regeneration of injured rat uterus.

Serious injuries of endometrium in women of reproductive age are often followed by uterine scar formation and a lack of functional endometrium predisposing to infertility or miscarriage. Bone marrow-derived mesenchymal stem cells (BM-MSCs) have shown great promise in clinical applications. In the present study, BM-MSCs loaded onto degradable collagen membranes were constructed. Collagen membranes provided 3-dimmensional architecture for the attachment, growth and migration of rat BM-MSCs and did not impair the expression of the stemness genes. We then investigated the effect of collagen/BM-MSCs constructs in the healing of severe uterine injury in rats (partial full thickness uterine excision). At four weeks after the transplantation of collagen/BM-MSCs constructs, BM-MSCs were mainly located to the basal membrane of regenerative endometrium. The wounded tissue adjacent to collagen/BM-MSCs constructs expressed higher level of bFGF, IGF-1, TGFβ1 and VEGF than the corresponding tissue in rats receiving collagen construct alone or in spontaneous regeneration group. Moreover, the collagen/BM-MSCs system increased proliferative abilities of uterine endometrial and muscular cells, facilitated microvasculature regeneration, and restored the ability of endometrium to receive the embryo and support its development to a viable stage. Our findings indicate that BM-MSCs may support uterine tissue regeneration.

An Increase of Treg Cells in the Peripheral Blood is Associated with a Better In Vitro Fertilization Treatment Outcome

The objective of this study was to determine whether there was any association between the peripheral blood CD4+ CD25+ Foxp3+ regulatory T cells (Treg cells) and implantation success in patients undergoing in vitro fertilization (IVF) treatment.

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

BACKGROUND Homeobox 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. OBJECTIVE The 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. METHODS The 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. RESULTS PCAF 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). CONCLUSIONS These 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.