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Oxidative stress-induced miR-27a targets the redox gene nuclear factor erythroid 2-related factor 2 in diabetic embryopathy.

ABSTRACT: BACKGROUND:Maternal diabetes induces neural tube defects, and oxidative stress is a causal factor for maternal diabetes-induced neural tube defects. The redox gene nuclear factor erythroid 2-related factor 2 is the master regulator of the cellular antioxidant system. OBJECTIVE:In this study, we aimed to determine whether maternal diabetes inhibits nuclear factor erythroid 2-related factor 2 expression and nuclear factor erythroid 2-related factor 2-controlled antioxidant genes through the redox-sensitive miR-27a. STUDY DESIGN:We used a well-established type 1 diabetic embryopathy mouse model induced by streptozotocin for our in vivo studies. Embryos at embryonic day 8.5 were harvested for analysis of nuclear factor erythroid 2-related factor 2, nuclear factor erythroid 2-related factor 2-controlled antioxidant genes, and miR-27a expression. To determine if mitigating oxidative stress inhibits the increase of miR-27a and the decrease of nuclear factor erythroid 2-related factor 2 expression, we induced diabetic embryopathy in superoxide dismutase 2 (mitochondrial-associated antioxidant gene)-overexpressing mice. This model exhibits reduced mitochondria reactive oxygen species even in the presence of hyperglycemia. To investigate the causal relationship between miR-27a and nuclear factor erythroid 2-related factor 2 in vitro, we examined C17.2 neural stem cells under normal and high-glucose conditions. RESULTS:We observed that the messenger RNA and protein levels of nuclear factor erythroid 2-related factor 2 were significantly decreased in embryos on embryonic day 8.5 from diabetic dams compared to those from nondiabetic dams. High-glucose also significantly decreased nuclear factor erythroid 2-related factor 2 expression in a dose- and time-dependent manner in cultured neural stem cells. Our data revealed that miR-27a was up-regulated in embryos on embryonic day 8.5 exposed to diabetes, and that high glucose increased miR-27a levels in a dose- and time-dependent manner in cultured neural stem cells. In addition, we found that a miR-27a inhibitor abrogated the inhibitory effect of high glucose on nuclear factor erythroid 2-related factor 2 expression, and a miR-27a mimic suppressed nuclear factor erythroid 2-related factor 2 expression in cultured neural stem cells. Furthermore, our data indicated that the nuclear factor erythroid 2-related factor 2-controlled antioxidant enzymes glutamate-cysteine ligase catalytic subunit, glutamate-cysteine ligase modifier subunit, and glutathione S-transferase A1 were down-regulated by maternal diabetes in embryos on embryonic day 8.5 and high glucose in cultured neural stem cells. Inhibiting miR-27a restored expression of glutamate-cysteine ligase catalytic subunit, glutamate-cysteine ligase modifier subunit, and glutathione S-transferase A1. Overexpressing superoxide dismutase 2 reversed the maternal diabetes-induced increase of miR-27a and suppression of nuclear factor erythroid 2-related factor 2 and nuclear factor erythroid 2-related factor 2-controlled antioxidant enzymes. CONCLUSION:Our study demonstrates that maternal diabetes-induced oxidative stress increases miR-27a, which, in turn, suppresses nuclear factor erythroid 2-related factor 2 and its responsive antioxidant enzymes, resulting in diabetic embryopathy.

SUBMITTER: Zhao Y

PROVIDER: S-EPMC5785788 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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Oxidative stress-induced miR-27a targets the redox gene nuclear factor erythroid 2-related factor 2 in diabetic embryopathy.

Zhao Yang Y Dong Daoyin D Reece E Albert EA Wang Ashley R AR Yang Peixin P

American journal of obstetrics and gynecology 20171101 1

<h4>Background</h4>Maternal diabetes induces neural tube defects, and oxidative stress is a causal factor for maternal diabetes-induced neural tube defects. The redox gene nuclear factor erythroid 2-related factor 2 is the master regulator of the cellular antioxidant system.<h4>Objective</h4>In this study, we aimed to determine whether maternal diabetes inhibits nuclear factor erythroid 2-related factor 2 expression and nuclear factor erythroid 2-related factor 2-controlled antioxidant genes thr ...[more]

PMID: 29100869

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Project description:Oxidative stress-induced vascular endothelial cell dysfunction serves an essential role in the initiation and development of atherosclerosis. Sulforaphane (SFN), a naturally occurring antioxidant, has previously demonstrated to exert protective effects on the endothelium against oxidative stress. However, further studies are required to determine its underlying molecular mechanism prior to clinical application. Accumulating evidence suggests that alterations in the microRNA (miRNA/miR)-34a/sirtuin-1 (SIRT1) axis occur with oxidative stress. Therefore, the present study aimed to investigate if SFN exerts a protective role against oxidative stress in vascular endothelial cells through regulation of the miR-34a/SIRT1 axis. Human umbilical vein endothelial cells (HUVECs) were treated with H2O2 in the presence or absence of SFN pretreatment. Cell viability and apoptosis were analyzed using CellTiter-Blue and flow cytometry, respectively. Reverse transcription-quantitative PCR and western blot analyses were performed to determine changes in the expression levels of miR-34a and SIRT1. The expression levels of miR-34a and SIRT1 were artificially regulated following transfection with miR-34a mimic and inhibitor or SIRT1expression plasmid and small interfering RNA, respectively. Subsequently, the effect of the expression changes of miR-34 and SIRT1 on oxidative stress-induced cell injury was investigated. Dual-luciferase reporter assay was used to confirm the targeted binding of miR-34a to SIRT1. SFN was found to ameliorate cellular damage caused by H2O2 and inhibited intracellular reactive oxygen species production. In addition, miR-34a upregulation was accompanied with reduced SIRT1 expression in HUVECs, following H2O2 treatment. miR-34a was revealed to directly target SIRT1 by binding to its 3'-untranslated region. Down-regulation of miR-34a and up-regulation of SIRT1 increased the survival of HUVECs under oxidative stress. Taken together, the results of the present study suggest that SFN may protect HUVECs from oxidative stress by inducing changes in the miR-34a/SIRT1 axis via upregulation of nuclear factor erythroid-2-related factor 2 expression.

Project description:BackgroundMaternal diabetes induces neural tube defects and stimulates the activity of the forkhead box O3 (Fox)O3a in the embryonic neuroepithelium. We previously demonstrated that deleting the FOXO3a gene ameliorates maternal diabetes-induced neural tube defects. Macroautophagy (hereafter referred to as "autophagy") is essential for neurulation. Rescuing autophagy suppressed by maternal diabetes in the developing neuroepithelium inhibits neural tube defect formation in diabetic pregnancy. This evidence suggests a possible link between FoxO3a and impaired autophagy in diabetic embryopathy.ObjectiveWe aimed to determine whether maternal diabetes suppresses autophagy through FoxO3a, and if the transcriptional activity of FoxO3a is required for the induction of diabetic embryopathy.Study designWe used a well-established type 1 diabetic embryopathy mouse model, in which diabetes was induced by streptozotocin, for our in vivo studies. To determine if FoxO3a mediates the inhibitory effect of maternal diabetes on autophagy in the developing neuroepithelium, we induced diabetic embryopathy in FOXO3a gene knockout mice and FoxO3a dominant negative transgenic mice. Embryos were harvested at embryonic day 8.5 to determine FoxO3a and autophagy activity and at embryonic day 10.5 for the presence of neural tube defects. We also examined the expression of autophagy-related genes. C17.2 neural stem cells were used for in vitro examination of the potential effects of FoxO3a on autophagy.ResultsDeletion of the FOXO3a gene restored the autophagy markers, lipidation of microtubule-associated protein 1A/1B-light chain 3I to light chain 3II, in neurulation stage embryos. Maternal diabetes decreased light chain 3I-positive puncta number in the neuroepithelium, which was restored by deleting FoxO3a. Maternal diabetes also decreased the expression of positive regulators of autophagy (Unc-51 like autophagy activating kinase 1, Coiled-coil myosin-like BCL2-interacting protein, and autophagy-related gene 5) and the negative regulator of autophagy, p62. FOXO3a gene deletion abrogated the dysregulation of autophagy genes. In vitro data showed that the constitutively active form of FoxO3a mimicked high glucose in repressing autophagy. In cells cultured under high-glucose conditions, overexpression of the dominant negative FoxO3a mutant blocked autophagy impairment. Dominant negative FoxO3a overexpression in the developing neuroepithelium restored autophagy and significantly reduced maternal diabetes-induced apoptosis and neural tube defects.ConclusionOur study revealed that diabetes-induced FoxO3a activation inhibited autophagy in the embryonic neuroepithelium. We also observed that FoxO3a transcriptional activity mediated the teratogenic effect of maternal diabetes because dominant negative FoxO3a prevents maternal diabetes-induced autophagy impairment and neural tube defect formation. Our findings suggest that autophagy activators could be therapeutically effective in treating maternal diabetes-induced neural tube defects.

Project description:Development is a complex and well-defined process characterized by rapid cell proliferation and apoptosis. At this stage in life, a developmentally young organism is more sensitive to toxicants as compared to an adult. In response to pro-oxidant exposure, members of the Cap'n'Collar (CNC) basic leucine zipper (b-ZIP) transcription factor family (including Nfe2 and Nfe2-related factors, Nrfs) activate the expression of genes whose protein products contribute to reduced toxicity. Here, we studied the role of the CNC protein, Nfe2, in the developmental response to pro-oxidant exposure in the zebrafish (Danio rerio). Following acute waterborne exposures to diquat or tert-buytlhydroperoxide (tBOOH) at one of three developmental stages, wildtype (WT) and nfe2 knockout (KO) embryos and larvae were morphologically scored and their transcriptomes sequenced. Early in development, KO animals suffered from hypochromia that was made more severe through exposure to pro-oxidants; this phenotype in the KO may be linked to decreased expression of alas2, a gene involved in heme synthesis. WT and KO eleutheroembryos and larvae were phenotypically equally affected by exposure to pro-oxidants, where tBOOH caused more pronounced phenotypes as compared to diquat. Comparing diquat and tBOOH exposed embryos relative to the WT untreated control, a greater number of genes were up-regulated in the tBOOH condition as compared to diquat (tBOOH: 304 vs diquat: 148), including those commonly found to be differentially regulated in the vertebrate oxidative stress response (OSR) (e.g. hsp70.2, txn1, and gsr). When comparing WT and KO across all treatments and times, there were 1170 genes that were differentially expressed, of which 33 are known targets of the Nrf proteins Nrf1 and Nrf2. More specifically, in animals exposed to pro-oxidants a total of 968 genes were differentially expressed between WT and KO across developmental time, representing pathways involved in coagulation, embryonic organ development, body fluid level regulation, erythrocyte differentiation, and oxidation-reduction, amongst others. The greatest number of genes that changed in expression between WT and KO occurred in animals exposed to diquat at 2h post fertilization (hpf). Across time and treatment, there were six genes (dhx40, cfap70, dnajb9b, slc35f4, spi-c, and gpr19) that were significantly up-regulated in KO compared to WT and four genes (fhad1, cyp4v7, nlrp12, and slc16a6a) that were significantly down-regulated. None of these genes have been previously identified as targets of Nfe2 or the Nrf family. These results demonstrate that the zebrafish Nfe2 may be a regulator of both primitive erythropoiesis and the OSR during development.

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Oxidative stress-induced miR-27a targets the redox gene nuclear factor erythroid 2-related factor 2 in diabetic embryopathy.

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Oxidative stress-induced miR-27a targets the redox gene nuclear factor erythroid 2-related factor 2 in diabetic embryopathy.

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