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. 2014 Dec 16;144(1):186–196. doi: 10.1093/toxsci/kfu271

The miR-322-TRAF3 Circuit Mediates the Pro-apoptotic Effect of High Glucose on Neural Stem Cells

Hui Gu *,†, Jingwen Yu *, Daoying Dong *, Qun Zhou ‡, Jian-Ying Wang §, Peixin Yang *,‡,1
PMCID: PMC4349142  PMID: 25516495

Abstract

Maternal diabetes increases the risk of neural tube defects (NTDs), and caspase-dependent apoptosis and gene dysregulation are implicated in this disease process. This study investigates the role of miR-322 and its putative target gene, TNF receptor-associated factor 3 (TRAF3), in high glucose-induced apoptosis. miR-322 and TRAF3 expression were assessed in embryos of nondiabetic and diabetic dams, and in neural stem cells under high glucose conditions. Maternal diabetes in vivo and high glucose in vitro significantly down-regulated miR-322 and up-regulated TRAF3 protein expression. Overexpression of the antioxidant enzyme, superoxide dismutase 1 (SOD1), or treatment with the SOD1 mimetic Tempol, abolished the effect of maternal diabetes or high glucose on miR-322 and TRAF3 expression, respectively. A miRNA target prediction algorithm reveals 2 miR-322 binding sites the 3′-untranslated region (UTR) of TRAF3 mRNA. A RNA pull-down assay using biotin-labeled miR-322 revealed that miR-322 interacted with the 3′-UTR of TRAF3 mRNA at one specific binding site. The miR-322 mimic or TRAF3 knockdown blocked high glucose-increased TRAF3 protein expression and apoptosis, whereas the miR-322 inhibitor mimicked the effect of high glucose leading to TRAF3 up-regulation and apoptosis. This study demonstrates that both maternal diabetes and high glucose negatively regulate miR-322 through oxidative stress. miR-322 interacts with the 3′-UTR of TRAF3 and represses its translation. The miR-322-TRAF3 pathway is implicated in high glucose-induced caspase activation and apoptosis.

Keywords: high glucose, apoptosis, microRNA-322, TRAF3, oxidative stress, neural stem cells

Pregestational maternal diabetes is a significant risk factor for neural tube defects (NTDs) in offspring (Correa et al., 2008). Previous studies have demonstrated that hyperglycemia caused by maternal diabetes increases the production of reactive oxygen species and suppresses endogenous antioxidant capacity leading to oxidative stress (Li et al., 2011, 2012, 2013; Sakamaki et al., 1999; Wentzel et al., 2008; Yang et al., 1997). Oxidative stress induces a set of pro-apoptotic kinase signaling intermediates, which influence gene expression that leads to programmed cell death of neural stem cells in the developing neuroepithelium (Wang et al., 2013, 2014; Yang et al., 2008a,b, 2007, 2013). Excess apoptosis in the neuroepithelium is the primary cause for NTD formation in diabetic pregnancies (Li et al., 2012, 2013; Xu et al., 2013; Yang et al., 2013). Although it is known that the pro-apoptotic kinase signaling downstream of oxidative stress induces pro-apoptotic gene transcription through the activation of transcription factors (Li et al., 2012, 2013; Wang et al., 2014; Yang et al., 2013), additional mechanisms, such as posttranscriptional mechanisms, may be involved in gene dysregulation downstream of oxidative stress in diabetic pregnancies.

MicroRNAs (miRNA) are a class of small non-coding RNAs that repress gene expression at the posttranscriptional level (Bartel, 2009). miRNAs bind to 3′-untranslated region (3′-UTR) of mRNAs through imperfect pairing to their seed sequences, and thereby degrading mRNAs, suppressing translation, or both (Lee et al., 2003). Through silencing the expression of their target genes, miRNAs exert pleiotropic functions by regulating cell apoptosis, proliferation, and differentiation (Xiao et al., 2011). Although some miRNAs suppress apoptosis, others promote apoptosis (Jovanovic and Hengartner, 2006; Ruan et al., 2014). Because apoptosis is a causal event in the induction of aberrant neural stem cell death in embryos exposed to high glucose of maternal diabetes (Li et al., 2012, 2013; Yang et al., 2013), it is reasoned that alterations of miRNA expression mediates the pro-apoptotic effects of high glucose.

There are multiple indirect evidence suggesting a critical role of miRNAs in maternal diabetes-induced apoptosis and consequent NTD formation. A dynamic change of miRNA expression is observed during neurulation in normal pregnancy (Mukhopadhyay et al., 2011). Altered miR expression has been detected in the maternal serum of human pregnancies affected by NTDs (Gu et al., 2012). Additionally, oxidative stress regulates miRNA expression (Magenta et al., 2011). Finally, high glucose directly modulates miRNA expression in cultured cells (Kantharidis et al., 2011). Exactly how hyperglycemia seen in maternal diabetes and high glucose in cultured embryos regulate the miRNA expression that contributes to apoptosis is unclear. Elucidating the role of miRNAs in hyperglycemia-induced apoptosis will add a new layer of regulators in the pathogenesis of diabetic embryopathy or diabetic complications in general.

miR-322, a miRNA abundantly expressed in many tissues, is located on the X chromosome (Griffiths-Jones et al., 2006). miR-322 has been shown to promote osteoblast differentiation (Gamez et al., 2013). During the differentiation of myoblast into myotubes, miR-322 together with miR-503 promote differentiation by inducing cell cycle arrest (Sarkar et al., 2010). A recent study reveals a critical role of miR-322 along with miR-503 in modulating TGF-β/Smad2 signaling and intestinal epithelial homeostasis, where they regulate intestinal epithelial cell apoptosis through inhibiting the translation of Smurf2, an E3 ubiquitin ligase for Smad2 degradation (Cao et al., 2014). In the current study, we found that maternal diabetes in vivo and high glucose in vitro inhibited miR-322 expression through oxidative stress. miR-322 interacts with the 3′-UTR of TNF receptor-associated factor 3 (TRAF3) mRNA and repressed TRAF3 translation. Under diabetic or high glucose conditions, miR-322 down-regulation causes TRAF3 overexpression, which leads to apoptosis. Moreover, a miR-322 mimic and a TRAF3 knockdown prevent high glucose-induced apoptosis, whereas a miR-322 inhibitor mimics the pro-apoptotic effects of high glucose. These findings indicate a critical role of the miR-322/TRAF3 regulatory pathway in the pathogenesis of neuroepithelial cell apoptosis and NTD formation in diabetic pregnancies.

MATERIALS AND METHODS

Animals and reagents

The procedures for animal use were approved by the University of Maryland School of Medicine Institutional Animal Care and Use Committee. Wild-type (WT) C57BL/6J mice were purchased from the Jackson Laboratory (Bar Harbor, Maine). Superoxide dismutase 1 (SOD1)-transgenic (Tg) mice in C57BL/6J background were revived from frozen embryos by the Jackson Laboratory (Stock number: 002298). Streptozotocin (STZ) from Sigma (St Louis, Missouri) was dissolved in sterile 0.1 M citrate buffer (pH 4.5). Sustained-release insulin pellets were purchased from Linpalant (Linshin, Canada).

Mouse models of diabetic embryopathy

Our mouse model of diabetic embryopathy has been described previously (Li et al., 2012, 2013; Yang et al., 2013). Briefly, 10-week old WT female mice were intravenously injected daily with 75 mg/kg STZ over 2 days to induce diabetes. Diabetes was defined as a 12-h fasting blood glucose level of more than or equal to 250 mg/dl. Insulin pellets were subcutaneously implanted in these diabetic mice to restore euglycemia prior to mating. Diabetic WT female mice were paired with SOD1-Tg male mice at 3 pm, and Day 0.5 (E0.5) of pregnancy was established by the presence of the vaginal plug at 8 am the next morning. On E5.5, insulin pellets were removed to ensure that the developing embryo would be exposed to a hyperglycemic environment during neurulation (E8-10.5). Nondiabetic WT female mice were treated with vehicle injection, and sham operation of insulin pellet implants/removal served as the nondiabetic controls. On E8.75, mice were euthanized and conceptuses were dissected out of the uteri for analyses.

Cell culture and chemicals

C17.2 mouse neural stem cells, originally obtained from European Collection of Cell Culture, were maintained in DMEM supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C in a humidified atmosphere of 5% CO2. Lipofectamine RNAiMAX (Invitrogen, Grand Island, New York) was used according to the manufacturer’s protocol for transfection of siRNA into the cells under 1% fetal bovine serum culture conditions. The mirVana miRNA mimic and the miRNA inhibitor of miR-322, the negative control (con) oligo (Cat. No. 4464058) for the miR-322 mimic, and the negative control oligo (Cat. No. 4464076) for the miR-322 inhibitor were purchased from Ambion (Austin, Texas). The SOD mimetic Tempol was purchased from Alexis Italia (Vinci, Florence, Italy). Biotin-labeled miRNA-322 was custom made by Dharmacon (Lafayette, Colorado).

Plasmid construction

The full-length TRAF3 coding region (CR) and 2 fractions (F1 and F2) in its 3′-UTR with 2 predicted miR-322 binding sites were amplified and subcloned into the pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega, Madison, Wisconsin) to generate the pmirGLO-Luc-TRAF3-CR and pmirGLO-TRAF3-F1/F2. The sequence and orientation of the fragments in the luciferase reporter were confirmed by DNA sequencing and enzymatic digestion. Luciferase activities were measured using the Dual-Luciferase Assay System, and were normalized by Renilla luciferase activities. All primer sequences for generating these constructs are provided in Supplementary Table 1.

RNA extraction and real-time PCR

Total RNA was isolated from embryos or cells using the mirVana miRNA isolation kit (Ambion, Life Technologies, Grand Island, New York) and reverse transcribed using the NCode VILO miRNA cDNA synthesis kit (Invitrogen, Life Technologies). RT-PCR for TRAF3, β-actin, miR-322, and small nuclear RNA U6 were performed using the Maxima SYBR Green/ROX qPCR Master Mix assay (Thermo Scientific, Rockford, Illinois). Real-time (RT)-PCR and subsequent calculations were performed by a StepOnePlus RT-PCR System (Applied Biosystem, Foster City, California). All primer sequences used in RT-PCR are provided in Supplementary Table 2.

Biotin-labeled miR-322 pulldown assay

Biotin-labeled negative control (Caenorhabditis elegans miR-67) or biotin-labeled miR-322 (CAGCAGCAAUUCAUGUUUUGGA-Biotin) was transfected into cells for 48 h, and then whole-cell lysate was collected. Cell lysates were mixed with streptavidin-coupled Dynabeads (Invitrogen, Carlsbad, California) and incubated at 4°C on rotator overnight. After the beads were washed thoroughly, the bead-bound RNA was isolated and subjected to reverse transcription followed by RT-PCR analyses. Input RNA was extracted and served as controls.

Immunoblotting

Equal amounts of protein from embryos or cells were resolved by SDS-PAGE and transferred onto Immunobilon-P membranes (Millipore, Billerica, Massachusetts). Membranes were incubated in 5% nonfat milk for 45 min and then incubated for 18 h at 4°C with the following primary antibodies at dilutions of 1:1000 in 5% nonfat milk: TRAF3, SOD1, and cleaved caspase 3 (Cell Signaling Technology, Danvers, Massachusetts). Membranes were then exposed to goat anti-rabbit or anti-mouse secondary antibodies. To confirm that equivalent amounts of protein were loaded among samples; membranes were stripped and probed with a mouse antibody against β-actin (Abcam). Signals were detected using the SuperSignal West Femto Maximum Sensitivity Substrate kit (Thermo Scientific). All experiments were repeated 3 times with the use of independently prepared tissue lysates. The dilutions and sources of antibodies in immunoblotting are provided in Supplementary Table 3.

TUNEL assay

The TUNEL assay was performed as previously described by using the In Situ Cell Death Detection Kit (EMD Millipore) (Li et al., 2012, 2013; Yang et al., 2013). Cells were seeded on 8-well Nunc Lab-Tek Chamber Slides (Sigma). After transfection and glucose treatment, cells were fixed with 4% paraformaldehyde in PBS and incubated with TUNEL reagent, counterstained with DAPI, and mounted with aqueous mounting medium (Sigma). TUNEL-positive cells in each well were counted. The percentage of apoptotic cells was calculated as the number of TUNEL-positive (apoptotic) cells, divided by the total number of cells.

Statistics

Data are presented as means ± SE. Three embryos from 3 different dams were used for immunoblotting and 6 embryos from 6 different dams were used for RT-PCR. One-way ANOVA or 2-way ANOVA (as indicated in figure legends) was performed using the SigmaStat 3.5 software, a Tukey’s multiple-comparison test or a T test was used to estimate the significance. Statistical significance was accepted when P < 0.05.

RESULTS

Hyperglycemia in vivo and High Glucose in vitro Decreases miR-322 Expression Through Oxidative Stress

To determine the involvement of miRNAs in the diabetic embryopathy, we carried out a microarray of global miRNA expression on embryos from diabetic and nondiabetic mice. We found that miR-322 was down-regulated 1.7-fold in embryos from diabetic dams compared with embryos from nondiabetic dams. To validate our finding, we examined the expression of miR-322 using RT-PCR. Maternal diabetes induces oxidative stress in the developing embryos (Yang et al., 2008a, 2010; Yang and Li, 2010). To explore whether oxidative stress mediates the inhibitory effect of maternal diabetes on miR-322 expression, we utilized embryos from nondiabetic WT and diabetic WT females that were mated with SOD1-Tg males.

The levels of miR-322 were significantly decreased in WT embryos exposed to maternal diabetes compared with WT embryos from nondiabetic dams (Fig. 1A). Because our previous studies have shown that SOD1 overexpression alleviates maternal diabetes-induced oxidative stress (Li et al., 2011; Wang et al., 2013; Weng et al., 2012), SOD1-overexpressing embryos were used to determine whether oxidative stress mediates the inhibitory effect of maternal diabetes on miR-322 expression. Indeed, SOD1 overexpression reversed maternal diabetes-suppressed miR-322 expression (Fig. 1A).

FIG. 1.

FIG. 1.

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High glucose in vivo and in vitro down-regulates miR-322 through oxidative stress. A, miR-322 levels in WT embryos from nondiabetic (ND) dams, WT, and SOD1 overexpressing embryos from diabetes mellitus (DM) dams mated with SOD1 transgenic males. Experiments were conducted using 6 embryos from 6 different dams (n = 6) per group. Asterisk (*) indicates significant differences (P < 0.05) compared with the other 2 groups. miR-322 levels in C17.2 neural stem cells cultured under normal glucose (5mM glucose) or high glucose (16.6, 25.0, and 33.3mM glucose) for 24 h (h) (B), 25mM glucose for 12, 24, and 48 h (C), 5 or 25mM glucose in the absence or presence of Tempol (100µM) for 24 h (D), 5mM glucose plus high mannitol (11.6, 20, and 28.3mM mannitol) for 24 h (E), and 5mM glucose plus 20mM mannitol for 12, 24, and 48 h (F). In B–F, values are mean ± SE of data from 3 separate experiments. In A, B, D, and E, 1-way ANOVA was used and in C and F, 2-way ANOVA was used for statistical analyses. Asterisk (*) indicates significant differences (P < 0.05) compared with the 5mM glucose group.

To reveal whether high glucose in vitro has a similar effect on miR-322 expression as maternal diabetes, C17.2 neural stem cells were cultured under normal glucose (5mM glucose) or high glucose (16.7, 25, and 33.3mM glucose). High glucose decreased miR-322 expression in a dose-dependent manner and the decline of miR-322 expression reached a plateau at 25mM glucose (Fig. 1B). 25mM glucose was comparable to the high blood glucose levels (average 26mM glucose) in the diabetic dams. A time course study of the effect of 25mM glucose revealed that miR-322 decreased at 12, 24, and 48 h with the deepest decline observed at 24 h (Fig. 1C). Treatment with the SOD1 mimetic Tempol abolished high glucose-decreased miR-322 expression (Fig. 1D). In addition, mannitol was used as an osmotic control of high glucose. When cells were treated with mannitol, high mannitol concentrations had no effect on miR-322 expression (Figs. 1E and 1F).

miR-322 Interacts with TRAF3 mRNA

Bioinformatic target prediction algorithm (miRanda) reveals that TRAF3 is a predicted target gene of miR-322, and there are 2 potential binding sites of miR-322 in the 3′-UTR of TRAF3 (Fig. 2A). To test whether TRAF3 is a true target of miR-322, we examined the association of miR-322 with TRAF3 mRNA by a RNA pulldown assay using the biotin-labeled miR-322. To assess the transfection efficiency, we examined the levels of miR-322 and small nuclear RNA U6 (which served as a control) after 48 h transfection. As shown in Figure 2B, cells transfected with the biotin-labeled miR-322 displayed elevated miR-322 levels but did not exhibit any changes in RNA U6 expression (Fig. 2C).

FIG. 2.

FIG. 2.

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miR-322 interacts with TRAF3 mRNA. A, Schematic representation of the TRAF3 mRNA depicting miR-322 binding sites in its 3′-UTR. Two predicted miR-322 binding sites in fraction 1 (F1) (position 2920–2941) and F2 (position 6797–6818) are located in the 3′-UTR of TRAF3 mRNA. B, miR-322 levels after 48 h biotin-miR-322 transfection. C, U6 RNA levels after 48 h biotin-miR-322 transfection. D, TRAF3 mRNAs levels in the materials pulled down by biotin-miR-322 and biotin-labeled negative control. E, Levels of input TRAF3 mRNA. Values are the means ± SE from 3 separate experiments. Asterisk (*) indicates significant differences (P < 0.05) in T tests compared with the control (Bio-con) group transfected with Biotin-labeled controls.

When the existence of TRAF3 mRNA in the cell lysates was examined, the levels of TRAF3 mRNA exhibited more than 16-fold more enrichment in the lysates from cells transfected with the biotin-labeled miR-322 than those from cells transfected with biotin-labeled scrambled control miRNA (Fig. 2D). In addition, increasing the levels of miR-322 by transfection with biotin-labeled miR-322 did not alter the total levels of TRAF3 mRNAs (Fig. 2E).

miR-322 Represses TRAF3 Translation

To define the functional consequences of miR-322/TRAF3-mRNA association, we used luciferase reporter constructs to examine whether miR-322 can directly regulate TRAF3 expression. miRNAs are able to repress gene expression by binding to seed site sequences located within the 3′-UTR of mRNA. Fractions of the CR of miR-322 mRNA or the specific binding sites (Fraction 1 [F1] and F2) of miR-322 in the TRAF3 3′-UTR were subcloned into the pmirGLO dual-luciferase miRNA target expression vector to generate CR-Luc, F1-Luc, and F2-Luc reporter constructs as indicated in Figure 3A. Ectopic miR-322 overexpression significantly decreased the luciferase activity of the F1-Luc reporter, but it failed to inhibit the activities of TRAF3 CR-Luc and F2-Luc reporter (Fig. 3A), indicating that miR-322 represses TRAF3 expression through its interaction with the F1 binding site in TRAF3 3′-UTR. To further verify the specific binding site of miR-322 in the TRAF3 3′-UTR, internal deletion mutation of the binding site located at the F1 of the TRAF3 3′-UTR was also performed, in which the nucleotides at positions 2920–2941 of the TRAF3 3′-UTR were eliminated (Fig. 3B). The mutant construct, F1-Luc-mut, lacking the possible binding site of miR-322 was refractory to the decrease in luciferase activity by miR-322 overexpression (Fig. 3B). Altogether, these results indicate that miR-322 represses TRAF3 expression through its interaction with one specific binding site of the TRAF3 3′-UTR.

FIG. 3.

FIG. 3.

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miR-322 inhibits TRAF3 translation. A, Levels of reporter activities as measured by analysis of TRAF3 CR, F1, or F2 (fractions of the TRAF3 3′-UTR encompass specific binding sites of miR-322) luciferase reporters after ectopic overexpression of miR-322. Left, Schematic of plasmids of different chimeric firefly luciferase TRAF3 reporters. BS, predicted miR-322-binding site. Right, Levels of activities of luciferase reporters containing TRAF3 CR, F1, or F2. Twenty-four hours after transfection with miR-322, cells were transfected with different TRAF3 luciferase reporter plasmids. Levels of firefly and Renilla luciferase activities were assayed 24 h later. Results were normalized to the Renilla luciferase activities and expressed as mean ± SE from 3 separate experiments (n = 3). B, Effect of miR-322-binding site (BS) deletion in F1 on luciferase reporter activities after ectopic miR-322 overexpression. C, miR-322 levels in cells transfected with the miR-322 mimic for 24 h. TRAF3 protein abundance (D) and mRNA levels (E) after 24 h miR-322 mimic overexpression. F, miR-322 levels in cells transfected with the miR-322 inhibitor. Effect of the miR-322 inhibitor on TRAF3 protein (G) and mRNA (H) expression. Experiments were repeated 3 times (n = 3). In B–H, 1-way ANOVA was used and in A, 2-way ANOVA was used for statistical analyses. Asterisk (*) indicates significant differences (P < 0.05) compared with cells transfected with the control oligos. The negative control (con) oligo (4464058) for the miR-322 mimic and the negative control oligo (4464076) for the miR-322 inhibitor were obtained from Ambion.

Next, the repression of TRAF3 expression by miR-322 was further characterized. In this study, miR-322 levels were increased remarkably by transfection with the miR-322 mimic (Fig. 3C). TRAF3 protein expression was significantly decreased by the miR-322 mimic (Fig. 3D), whereas no significant differences were found in TRAF3 mRNA levels (Fig. 3E). On the other hand, when miR-322 levels were decreased by transfection with the miR-322 inhibitor (Fig. 3F), TRAF3 protein levels increased accordingly (Fig. 3G), although there were no significant changes in TRAF3 mRNA levels (Fig. 3H).

High Glucose Up-regulates TRAF3 Expression Through miR-322

Since high glucose down-regulates miR-322, we sought to determine whether high glucose regulates TRAF3 expression. TRAF3 protein levels were significantly increased in WT embryos exposed to maternal diabetes compared with embryos from nondiabetic WT mice (Fig. 4A). SOD1-overexpressing embryos had significantly lower levels of TRAF3 than those in WT embryos from the same group of diabetic WT dams mated with SOD1-Tg males (Fig. 4A). However, maternal diabetes did not affect TRAF3 mRNA expression (Fig. 4B). The results were consistent when we extended our observations to in vitro experiments. The up-regulation of protein but not mRNA levels of TRAF3 was observed in cells treated with 25mM glucose (Figs. 4C and 4D).

FIG. 4.

FIG. 4.

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High glucose up-regulates TRAF3 protein expression by suppressing miR-322. Levels of TRAF3 protein (A) and mRNA (B) in WT embryos from nondiabetic (ND) dams, WT, and SOD1 overexpressing from diabetes mellitus (DM) dams mated with SOD1 transgenic males. Experiments were conducted using embryos from 3 different dams (n = 3) per group and the quantification of the data was shown in the bar graph. Asterisk (*) indicates significant differences (P < 0.05) compared with the other 2 groups. Changes in TRAF3 protein expression (C) and mRNA expression (D) when cells were cultured under 5 or 25mM glucose for 24 h. E, TRAF3 protein abundance in cells transfected with the control oligo or the miR-322 mimic under normal or high glucose conditions. Experiments were repeated 3 times (n = 3) and the quantification of the data was shown in the bar graph. F, TRAF3 protein levels in cells transfected with the control oligo or the miR-322 inhibitor under normal or high glucose conditions. Experiments were repeated 3 times (n = 3) and the quantification of the data was shown in the bar graph. In A, E, and F, 1-way ANOVA was used and in B, C, and D, T tests were used for statistical analyses. Asterisk (*) indicates significant differences (P < 0.05) compared with the 5mM glucose group. Hash (#) indicates significant differences (P < 0.05) compared with the control group.

Furthermore, to explore whether miR-322 mediates the stimulative effect of high glucose on TRAF3 protein expression, cells were transfected with the miR-322 mimic under normal glucose (5mM) or high glucose (25mM) conditions. The miR-322 mimic blocked high glucose-increased TRAF3 (Fig. 4E). In contrast, the miR-322 inhibitor mimicked the stimulatory effect of high glucose on TRAF3 protein expression (Fig. 4F). Thus, these findings support that high glucose up-regulates TRAF3 protein expression through repression of miR-322.

High Glucose Induces Apoptosis Through miR-322 Down-regulation and TRAF3 Up-regulation

To define the biological effect of high glucose-decreased miR-322 expression and -increased TRAF3, we assessed caspase cleavage and the number of apoptotic cells. When cells were exposed to high glucose, apoptotic cells were robustly present whereas the miR-322 mimic protected cells from high glucose-induced apoptosis (Fig. 5A). High glucose increased the abundance of cleaved caspase 3 and the miR-322 mimic blocked the increase in caspase 3 cleavage (Fig. 5B). TRAF3 siRNA effectively reduced TRAF3 protein expression by more than 70% (Fig. 5C). Similar to the effect of the miR-322 mimic, TRAF3 knockdown abolished high glucose-induced caspase 3 cleavage (Fig. 5D) and reduced the number of apoptotic cells (Fig. 5E). These data suggest that miR-322 protects cells from high glucose-induced apoptosis by suppressing TRAF3 expression.

FIG. 5.

FIG. 5.

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High glucose induces apoptosis by down-regulating miR-322 and up-regulating TRAF3 protein. A, Representative images of the TUNEL assay. Cells were transfected with the control oligo or the miR-322 inhibitor under normal or high glucose conditions. Apoptotic cells were labeled by the TUNEL reagents and all cell nuclei were stained by DAPI. B, cleaved Caspase 3 levels after cells were transfected with the control oligo or the miR-322 inhibitor under normal or high glucose conditions. C, TRAF3 protein level after cells were transfected with control siRNA or TRAF3 siRNA at different concentrations. Experiments were repeated three times (n = 3) and the quantification of the data were shown in the bar graph. * indicate significant differences (P < 0.05) compared with the control group. D, cleaved Caspase 3 levels after cells were transfected with the control siRNA (35 nM) or TRAF3 siRNA (35 nM) under normal or high glucose conditions. E, Representative images of the TUNEL assay. Cells were transfected with the control siRNA (35 nM) or TRAF3 siRNA (35 nM) under normal or high glucose conditions. Apoptotic cells were labeled by the TUNEL reagents and all cell nuclei were stained by DAPI. In A, B, C, D, experiments were repeated three times (n = 3). The bars in A and E were 60 mM. * indicate significant differences (P < 0.05) in one-way ANOVA analyses compared to the other two groups.

DISCUSSION

Hyperglycemia (high glucose) of diabetes alters miRNA expression in specific tissues (Kantharidis et al., 2011). High glucose in vitro recapitulates the effects of diabetes in vivo in terms of modulating miRNA expression (Kantharidis et al., 2011). High glucose regulates miRNA expression in a tissue- and cell-specific manner by up-regulating one group of miRNAs, whereas down-regulating another (Kantharidis et al., 2011). High glucose-induced apoptosis is the main causal factor in the etiology of many diabetic complications (Allen et al., 2005), including NTD formation caused by maternal diabetes in vivo and high glucose in vitro-induced neural stem cell apoptosis (Yang et al., 2013). Previous studies have demonstrated that miRNAs mediate the pro-apoptotic effect of high glucose (Kantharidis et al., 2011). In diabetic nephropathy, high glucose-induced miR-29c triggers podocyte apoptosis and increases extracellular matrix protein accumulation (Long et al., 2011). High glucose-increased miR-1 induces cardiomyocyte apoptosis through inhibiting a pro-survival factor, insulin-like growth factor 1 (Yu et al., 2008). The present study reveals a new paradigm in which high glucose-suppressed miR-322 expression causes an elevation of the pro-apoptotic factor TRAF3 that contributes to neural stem cell apoptosis.

High glucose induces oxidative stress in the developing embryo by enhancing reactive oxygen species production and blunting cellular antioxidant capacities (Yang et al., 2008a). Antioxidant treatments can prevent NTD formation in vivo and in vitro (Yang and Li, 2010), supporting our central hypothesis that oxidative stress causes diabetic embryopathy. SOD1 overexpression in transgenic mice, which is known to diminish oxidative stress in embryos exposed to diabetes (Li et al., 2011) restores miR-322 expression that is repressed by diabetes. The cell-permeable SOD1 mimetic Tempol, which scavenges superoxide (Ishizuka et al., 2011), also restores miR-322 expression. Collectively, these findings indicate that oxidative stress is the cause of miR-322 down-regulation. SOD1 overexpression in vivo blocks maternal diabetes-increased TRAF3 protein expression.

We recently revealed a set of kinase signaling intermediates downstream of high glucose including apoptosis signal-regulating kinase 1 (ASK1) and c-Jun-N-terminal kinase 1/2 (JNK1/2), which activates a group of transcription factors (Yang et al., 2013). Because there are virtually no data available regarding transcriptional regulation of miR-322 expression, future studies aim to explore whether the kinase-activated transcription factors are involved in high glucose-induced miR-322 down-regulation.

miR-322 is clustered with miR-503 (Griffiths-Jones et al., 2006), and the primary functions of miR-322 and miR-503 seem to be promoting cell differentiation and inhibiting cell proliferation (Sarkar et al., 2010). miR-322 and miR-503 are induced during muscle differentiation, and their overexpression induces cell cycle arrest (Sarkar et al., 2010). In vascular smooth muscle cells, miR-322 is up-regulated after vascular injury and miR-322 overexpression inhibits cell proliferation and migration (Merlet et al., 2013). In human cancer cells, miR-424, the human ortholog of miR-322, along with miR-503 is repressed, and introducing these 2 miRNAs to cancer cells slows down tumor progression (Oneyama et al., 2013). Our study demonstrated a new function of miR-322 in promoting neural stem cell survival by repressing TRAF3 translation. Multiple approaches were used to define TRAF3 mRNA as the regulatory target of miR-322. Biotin-labeled miR-322 is enriched in TRAF3 mRNA. Luciferase reporter assays driven by different 3′-UTR fragments of TRAF3 RNA determine one functional miR-322 binding site in TRAF3 mRNA. Furthermore, the miR-322 mimic reduces TRAF3 protein expression, whereas the miR-322 inhibitor up-regulates TRAF3 protein expression. miR-322 mediates the pro-apoptotic effect of high glucose in neural stem cells because the miR-322 mimic abrogates high glucose-increased TRAF3 expression, caspase3 cleavage, and apoptosis.

TRAF3, a member of the TRAF3 family, was initially discovered as an adaptor protein that binds to 2 TNFRs, CD40 and LMP-1 (Cheng et al., 1995). A TRAF3 mutant lacking its amino-terminal domain, which displaces endogenous TRAF3 from lymphotoxin beta receptor (LT-βR), inhibits LT-β-induced apoptosis (VanArsdale et al., 1997), suggesting a pro-apoptotic function of TRAF3. A direct role for TRAF3 in apoptosis is likely because CD40-induced apoptosis is associated with TRAF3 protein up-regulation, and knocking down TRAF3 abrogates pro-apoptotic JNK1/2 activation and CD40-induced apoptosis (Georgopoulos et al., 2006). TRAF3 also interacts with the JNK1/2 upstream kinase, ASK1, in mediating the pro-apoptotic signal of LT-βR (Chen et al., 2003). Because we previously revealed that the pro-apoptotic ASK1-JNK1/2 pathway plays a causal role in maternal diabetes- and high glucose-induced neural stem cell apoptosis, which leads to NTD formation (Li et al., 2012, 2013; Yang et al., 2013), it is possible that TRAF3 cooperates with the ASK1-JNK1/2 pathway in the induction of cell death under high glucose conditions.

In summary, high glucose in vivo and in vitro cause oxidative stress which, in turn, represses miR-322 expression and increases the pro-apoptotic TRAF3 protein abundance. TRAF3 mRNA is a new target of miR-322, and appears to be essential for high glucose-induced neural stem cell apoptosis. miR-322 inhibits TRAF3 mRNA translation but does not affect its stability. Overexpressing miR-322 by the miR-322 mimic, as well as siRNA knocking down TRAF3 rescue neural stem cells from high glucose-induced caspase activation and apoptosis. Our study unravels the miR-322-TRAF3 circuit in high glucose-induced cell apoptosis, and provides new insights into our understanding of the mechanisms underlying apoptosis-induced diabetic complications, particularly, maternal diabetes-induced NTD formation in the developing embryo.

SUPPLEMENTARY DATA

Supplementary data are available online at http://toxsci.oxfordjournals.org/.

FUNDING

The National Institutes of Health R01DK083243, R01DK101972, R56DK095380, R01DK103024, and the Basic Science Award (1-13-BS-220), American Diabetes Association.

Supplementary Material

Supplementary Data
supp_144_1_186__index.html (938B, html)

ACKNOWLEDGMENTS

The authors thank the support from the Office of Dietary Supplements, National Institute of Health (NIH) for providing financial support. They are grateful to Dr Julie Wu at the University of Maryland School of Medicine for critical reading and editing assistance.

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