RESUMO
Inflammatory cytokines, including tumor necrosis factor-α (TNFα), were elevated in patients with cardiovascular diseases and are also considered as crucial factors in the pathogenesis of preeclampsia; however, the underlying pathogenic mechanism has not been clearly elucidated. This study provides novel evidence that TNFα leads to endothelial dysfunction associated with hypertension and vascular remodeling in preeclampsia through down-regulation of endothelial nitric-oxide synthase (eNOS) by NF-κB-dependent biogenesis of microRNA (miR)-31-5p, which targets eNOS mRNA. In this study, we found that miR-31-5p was up-regulated in sera from patients with preeclampsia and in human endothelial cells treated with TNFα. TNFα-mediated induction of miR-31-5p was blocked by an NF-κB inhibitor and NF-κB p65 knockdown but not by mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase inhibitors, indicating that NF-κB is essential for biogenesis of miR-31-5p. The treatment of human endothelial cells with TNFα or miR-31-5p mimics decreased endothelial nitric-oxide synthase (eNOS) mRNA stability without affecting eNOS promoter activity, resulting in inhibition of eNOS expression and NO/cGMP production through blocking of the functional activity of the eNOS mRNA 3'-UTR. Moreover, TNFα and miR-31-5p mimic evoked endothelial dysfunction associated with defects in angiogenesis, trophoblastic invasion, and vasorelaxation in an ex vivo cultured model of human placental arterial vessels, which are typical features of preeclampsia. These results suggest that NF-κB-responsive miR-31-5p elicits endothelial dysfunction, hypertension, and vascular remodeling via post-transcriptional down-regulation of eNOS and is a molecular risk factor in the pathogenesis and development of preeclampsia.
Assuntos
Células Endoteliais/fisiologia , MicroRNAs/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Pré-Eclâmpsia/metabolismo , Regiões 3' não Traduzidas/genética , Animais , Artérias/efeitos dos fármacos , Regulação para Baixo , Células Endoteliais/efeitos dos fármacos , Feminino , Técnicas de Silenciamento de Genes , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos Endogâmicos C57BL , MicroRNAs/farmacologia , Subunidade p50 de NF-kappa B/genética , Subunidade p50 de NF-kappa B/metabolismo , Neovascularização Fisiológica , Placenta/irrigação sanguínea , Placenta/efeitos dos fármacos , Pré-Eclâmpsia/genética , Gravidez , Trofoblastos/fisiologia , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Chronic thromboembolic pulmonary hypertension (CTEPH) is a leading cause of pulmonary hypertension. The present study investigated the mechanisms of long noncoding RNA growth arrestspecific transcript 5 (GAS5) on spermidine (SP)induced autophagy. Pulmonary artery endothelial cells (PAECs) were collected from patients with CTEPH and the rat model. Immunofluorescence, Western blots, reverse transcriptionquantitative polymerase chain reaction, bioinformatics, rapid amplification of cDNA ends assays, luciferase reporter assays, RNAbinding protein immunoprecipitation assays, GFPLC3 adenoviruses, tfLC3 assays and transmission electron microscopy were performed. The results revealed that SPinduced autophagy increased GAS5 in PAECs. The upregulation of GAS5 enhanced and the downregulation of GAS5 reversed the roles of SP in PAECs. Furthermore, GAS5 promoted SPinduced autophagy in PAECs by targeting miRNA315p. The miRNA315p mimic suppressed and the inhibitor promoted SPinduced autophagy. Furthermore, NAcetyltransferase 8 Like (NAT8L) was a target gene of miRNA315p and knockdown of NAT8L inhibited the autophagic levels of PAECs. In vivo, SP treatment decreased miRNA315p and increased NAT8L levels, which was reversed by the knockdown of GAS5. The downregulation of GAS5 abolished the stimulatory role of SP in PAECs of CTEPH rats. In conclusion, GAS5 promoted SPinduced autophagy through miRNA315p/NAT8L signaling pathways in vitro and in vivo and GAS5 may be a promising molecular marker for therapies of CTEPH.1.
Assuntos
Acetiltransferases , Hipertensão Pulmonar , MicroRNAs , RNA Longo não Codificante , Acetiltransferases/genética , Acetiltransferases/metabolismo , Animais , Autofagia , Células Endoteliais/metabolismo , MicroRNAs/metabolismo , Artéria Pulmonar/metabolismo , RNA Longo não Codificante/metabolismo , Ratos , EspermidinaRESUMO
Several lines of evidence highlight the important application of human spermatogonial stem cells (SSCs) in translational medicine. The fate decisions of SSCs are mainly mediated by genetic and epigenetic factors. We have recently demonstrated that PAK1 regulates the proliferation, DNA synthesis, and early apoptosis of human SSCs through the PDK1/KDR/ZNF367 and ERK1/2 and AKT pathway. However, the underlying epigenetic mechanism of PAK1 in human SSCs remains unknown. In this study, we found that the level of miRNA-31-5p was elevated by PAK1 knockdown. CCK-8, PCNA, and 5-ethynyl-2'-deoxyuridine (EDU) assays revealed that miRNA-31-5p mimics inhibited cell proliferation and DNA synthesis of human SSCs. Annexin V/propidium iodide (PI) staining and flow cytometry showed that miRNA-31-5p increased the early and late apoptosis of human SSCs. Furthermore, JAZF1 was predicted and verified as a target of miRNA-31-5p, and the three-dimensional (3D) structure model of JAZF1 protein was illustrated. JAZF1 silencing led to a reduction of cell proliferation and DNA synthesis as well as an enhancement of the early and late apoptosis of human SSCs. Finally, miRNA-31-5p mimics decreased the level of cyclin A2 rather than cyclin D1 or cyclin E1, and JAZF1 knockdown led to the reduction of cyclin A2 in human SSCs. Collectively, miRNA-31-5p regulates the proliferation, DNA synthesis, and apoptosis of human SSCs by the PAK1-JAZF1-cyclin A2 pathway. This study thus offers a novel insight into the molecular mechanisms underlying the fate determinations of human SSCs and might provide novel targets for molecular therapy of male infertility.