Zhenyu Diao

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.

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.

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.

MicroRNA-181a is involved in the regulation of human endometrial stromal cell decidualization by inhibiting Krüppel-like factor 12

BackgroundThe transformation of endometrium into decidua is essential for normal implantation of the blastocyst. However, the post-transcriptional regulation and the miRNAs involved in decidualization remain poorly understood. Here, we examined microRNA-181a (miR-181a) expression in decidualized human endometrial stromal cell (hESC). In addition, we investigated the functional effect of miR-181a on hESC decidualization in vitro.MethodsQuantitative real-time PCR (qRT-PCR) was used to detect the profile of miR-181a in decidualized hESC. qRT-PCR, enzyme-linked fluorescent assay, and immunofluorescence assay were performed to investigate decidualization marker genes’ expression after enhancing or inhibition of miR-181a expression in hESC. Luciferase reporter assay, western blotting, qRT-PCR, and immunofluorescence assay were carried out to identify the relationship between miR-181a and Krüppel-like factor 12 (KLF12).ResultsmiR-181a expression levels increased dramatically in hESC treated with 8-Br-cAMP and MPA. Increased miR-181a expression promoted hESC decidualization-related gene expression and morphological transformation; conversely, inhibition of miR-181a expression compromised hESC decidualization in vitro. Further analysis confirmed that miR-181a interacted with the 3′ untranslated region of the transcription factor KLF12 and down-regulated KLF12 at the transcriptional and translational levels. KLF12 overexpression abolished miR-181a-induced decidualization.ConclusionsOur findings suggest that miR-181a plays a functionally important role in human endometrial stromal cell decidualization in vitro by inhibiting KLF12.

NUR77 inhibits the expression of TIMP2 and increases the migration and invasion of HTR-8/SVneo cells induced by CYR61

OBJECTIVE To investigate the function and mechanism of CYR61 on the migration and invasion of the trophoblast cell line, HTR-8/SVneo cells. STUDY DESIGN The mRNA and protein levels of NUR77 in the placentas of normal and preeclampsia (PE) women were evaluated using real-time PCR and Western blot, respectively. Paraffin-embedded tissues were processed for localization of NUR77 protein in placental villus by immunohistochemistry. HTR-8/SVneo cells were cultured in the presence of CYR61, Ad-NUR77 or a small interfering RNA for NUR77 (Ad-sinur77). The expression of NUR77 in the HTR-8/SVneo cells was detected and the effects of CYR61 on the migration and invasion of HTR-8/SVneo cells were assessed in wound-healing and transwell experiments, respectively. Gelatin zymography was used to measure the MMP2 release in HTR-8/SVneo cells. RESULTS NUR77 is significantly decreased in the placenta of women with PE compared with the levels during a normal pregnancy. CYR61 can significantly increase the expression of NUR77 in HTR-8/SVneo cells. CYR61, as well as NUR77, can promote HTR-8/SVneo cells migration and invasion, which can be blocked by Ad-sinur77. Both CYR61 and Ad-nur77 reduced the mRNA expression of TIMP2 in HTR-8/SVneo cells. CONCLUSIONS CYR61 may promote HTR-8/SVneo cells migration and invasion through the upregulation of NUR77, leading to the increase of MMP2 release and the downregulation of TIMP2 expression.

CYR61 Modulates the Vascular Endothelial Growth Factor C Expression of Decidual NK Cells Via PI3K/AKT Pathway

Citation Zhang X, Ding L, Diao Z, Yan G, Sun H, Hu Y. CYR61 modulates the vascular endothelial growth factor C expression of decidual NK cells via PI3K/AKT pathway. Am J Reprod Immunol 2012; 67: 216–223

Tripterygium Glycosides Impairs the Proliferation of Granulosa Cells and Decreases the Reproductive Outcomes in Female Rats

This study was carried out to investigate the impact of tripterygium glycosides (TGs) on ovarian function of female rats in vitro and in vivo. In vitro studies showed that TG induced cells decrease at G1 phase and inhibited cell proliferation in rat granulosa cells. In vivo, female rats were intragastrically administered with TG at the dose of 60 mg/kg/day for consecutive 50 days. TG caused a prolonged estrous cycle, and a significant reduction in ovarian index, serum E2 level, and numbers of secondary and antral follicles (p < 0.05) in these rats. A significant reduction of viable embryos was demonstrated in TG-treated female rats after mating (p < 0.01). Further, we observed observed the reduced expression level of TGF-β1 after TG treatment in vitro and in vivo. Moreover, the expression of Smad2 and AKT was also decreased after TG treatment. These results suggest that TG can impair ovarian function through Smads-mediated TGF-β1 signal pathway.

miR-133b Reverses the Hydrosalpinx-induced Impairment of Embryo Attachment Through Down-regulation of SGK1

CONTEXT Hydrosalpinx impairs endometrial receptivity and embryo implantation. However, the exact underlying mechanism remains elusive. OBJECTIVE This study aimed to explore how an miR-133b-mediated mechanism controls endometrial receptivity and embryo attachment in the endometrium of women with hydrosalpinx. DESIGN, SETTING, PATIENTS, AND INTERVENTIONS Ishikawa cells were treated with hydrosalpinx fluid (HF) from infertile patients and cultured for in vitro analysis. The attachment rates of BeWo spheroids and mouse embryos to Ishikawa cells were assayed. PRIMARY OUTCOME MEASURE miR-133b, serum and glucocorticoid-regulated kinase 1 (SGK1), and homeobox A10 (HOXA10) expression levels were evaluated by quantitative real-time PCR and Western blot assays. RESULTS The expression of miR-133b and HOXA10 was significantly down-regulated, whereas the miR-133b target gene SGK1 was up-regulated in mid-secretory endometrial tissues of women with hydrosalpinx and in HF-treated Ishikawa cells. Moreover, hydrosalpinx inhibited miR-133b expression through the activation of nuclear factor κB/p65, and SGK1 decreased miR-133b-induced HOXA10 expression by phosphorylating cAMP responsive element binding protein in Ishikawa cells. Our results also showed that miR-133b and HOXA10 contributed to BeWo spheroid adhesiveness, whereas SGK1 inhibited BeWo spheroid attachment to Ishikawa cells. Importantly, miR-133b overexpression reversed the HF-mediated impairment of embryo attachment in vitro. CONCLUSIONS miR-133b directly targets SGK1 to reverse the hydrosalpinx-induced down-regulation of HOXA10 and to attenuate the impairment of embryo attachment in vitro.

Krüppel-like factor 12 is a novel negative regulator of forkhead box O1 expression: a potential role in impaired decidualization

BackgroundDecidualization is a prerequisite for successful implantation and the establishment of pregnancy. Krüppel-like factor 12 (KLF12) is a negative regulator of endometrial decidualization in vitro. We investigated whether KLF12 was associated with impaired decidualization under conditions of repeated implantation failure (RIF).MethodsUterine tissues were collected from a mouse model of early pregnancy and artificial decidualization for immunohistochemistry, Western blot and real-time PCR analysis. Reporter gene assays, chromatin immunoprecipitation-PCR and avidin-biotin conjugate DNA precipitation assays were performed to analyze the transcriptional regulation of forkhead box O1 (FOXO1) by KLF12. Furthermore, the protein levels of KLF12 and FOXO1 in patients with RIF were analyzed by Western blot and immunohistochemistry.ResultsKLF12 led to defective implantation and decidualization in the mouse uterine model of early pregnancy and artificial decidualization by directly binding to the FOXO1 promoter region and inhibiting its expression in human endometrial stromal cells. Elevated KLF12 expression was accompanied by decreased FOXO1 expression in the endometria of patients with RIF.ConclusionsAs a novel regulator, KLF12 predominantly controls uterine endometrial differentiation during early pregnancy and leads to implantation failure.

The clinical application of NGS-based SNP haplotyping for PGD of Hb H disease

ABSTRACT This study investigated the usefulness of next-generation sequencing (NGS)-based single nucleotide polymorphism (SNP) haplotyping for preimplantation genetic diagnosis (PGD) of hemoglobin H (Hb H) disease. Multiple displacement amplification (MDA) was used for whole genome amplification (WGA) of biopsied trophectoderm (TE) cells. Gap-PCR and NGS-based SNP haplotyping was used to distinguish the two genotypes of -α3.7/αα and –SEA/αα for PGD of Hb H disease. One out of the ten blastocysts (B11) was successfully diagnosed as genotype -α3.7/αα by Gap-PCR, whereas the others revealed allele dropout (ADO) (B1, B2, B4, B5, B7, B8, B12, and B15) or amplification failure (B10). However, NGS-based SNP haplotyping successfully diagnosed the -α3.7/αα and –SEA/αα genotypes from the MDA products of the biopsied TE cells. The haplotyping result showed that B4, B7, B8, B10, B11, B12, and B15 were carriers of the -α3.7 deletion (-α3.7/αα), whereas B1, B2, and B5 were carriers of the –SEA deletion (–SEA/αα). A blastocyst (B11) was transferred into the uterus in a subsequent frozen embryo transfer (FET) cycle after PGD. A healthy infant with a -α3.7/αα genotype weighing 2,800 g was born by cesarean section at the 38th week of gestation. This result indicates that NGS-based SNP haplotyping is a valid screening tool for the PGD of Hb H disease.