ASK1 modulates the expression of microRNA Let7A in microglia under high glucose in vitro condition

Hyperglycemia results in oxidative stress and leads to neuronal apoptosis in the brain. Diabetes studies show that microglia participate in the progression of neuropathogenesis through their involvement in inflammation in vivo and in vitro. In high-glucose-induced inflammation, apoptosis signal regulating kinase 1 (ASK1) triggers the release of apoptosis cytokines and apoptotic gene expression. MicroRNA-Let7A (miR-Let7A) is reported to be a regulator of inflammation. In the present study, we investigated whether miR-Let7A regulates the function of microglia by controlling ASK1 in response to high-glucose-induced oxidative stress. We performed reverse transcription (RT) polymerase chain reaction, Taqman assay, real-time polymerase chain reaction, and immunocytochemistry to confirm the alteration of microglia function. Our results show that miR-Let7A is associated with the activation of ASK1 and the expression of anti-inflammatory cytokine (interleukin (IL)-10) and Mycs (c-Myc and N-Myc). Thus, the relationship between Let-7A and ASK1 could be a novel target for enhancing the beneficial function of microglia in central nervous system (CNS) disorders.


miRNA Transfection
BV2 microglia were treated with miR-Let7A mimic or anti-miR-Let7A. Let-7A mimic (Let-7A precursor molecules are considered to be processed into mature miRNA by mimicking the Let-7A natural shearing process) and anti-Let-7A (Let7A inhibitor) were purchased from Ambion (Austin, TX, USA). For the transfection of RNA duplexes, a 20 nM final concentration of Let-7A miRNA mimic or Let-7A inhibitor was mixed with lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) in Opti-MEM medium and incubated at room temperature for 10 min. The mixture was added to cells in 6-well plates. After cells reached 60% confluence, they were harvested for total protein or RNA extraction. ASK1 inhibitor (NQDI-1) was purchased from Tocris Bioscience (Bristol, UK). BV2 microglial cells were pretreated with ASK1 inhibitor (600 nM) to inhibit ASK1 activation 2 h before high-glucose injury.

Cell Viability Assay
BV2 microglia (2 × 10 5 cells/ml) were seeded in 96-well plates to examine the effects of all experimental treatments. Cells were then rinsed with phosphate-buffered saline (PBS), and culture medium was replaced with serum-free medium. Next, 100 µl 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma, St. Louis, MO, USA) solution (5 mg/ml in PBS) was added per well. After 1 h of incubation, the medium was removed, and dimethyl sulfoxide was added to solubilize the purple formazan product of the MTT reaction. The supernatant from each well was measured using an ELISA plate reader at a wavelength of 570 nm with background subtraction at 650 nm. All experiments were repeated at least six times. Cell viability was not considered 100% for any treatment group. Cell viability was expressed as a percentage relative to the control group value.

Reverse Transcription Polymerase Chain Reaction (RT-PCR)
To examine the expression of IL-10, c-Myc, and N-Myc in BV2 microglia, RT-PCR was performed using each primer. Briefly, samples were lysed with Trizol reagent (Invitrogen, Carlsbad, CA, USA), and total RNA was extracted according to the manufacturer's protocol. cDNA synthesis from mRNA and sample normalization were performed. PCR was performed using the following thermal cycling conditions: 95 • C for 10 min; 35 cycles of denaturing at 95 • C for 15 s, annealing at 70 • C for 30 s, elongation at 72 • C for 30 s; final extension at 72 • C for 10 min; and holding at 4 • C. PCR was performed using the following primers (5 ′ -3 ′ ); IL-10 (

Quantitative Real-time PCR
To examine the amount of TNF-α, IL-6, IL-10, and ASK1 mRNA in BV2 cells under high-glucose conditions, quantitative real-time PCR was performed using each primer. Total cellular RNA was extracted from BV2 microglia using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. RNA was mixed with One Step SYBR R Prime Script TM RT-PCR Kit II (Takara, Otsu, Shiga, Japan) and specific primers in a total reaction volume of 20 µl. PCR was performed using the following primers (5 ′ -3 ′ ); TNF-α (  (Popivanova et al., 2008). GAPDH was amplified as an internal control. The Ct value for GAPDH was subtracted from the Ct values for each gene ( Ct). The Ct values of treated cells were compared with those of untreated cells.

Taqman Assay
Total RNA was extracted from cells using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. For quantitative analysis of miR-Let7A, reverse transcription (RT) was performed using Taqman Micro RNA RT kit (Takara, Otsu, Shiga, Japan) according to the manufacturer's instructions with 10 ng total RNA. PCR reactions were then performed according to manufacturer's instructions to quantify the expression level of miR-Let7A using Taqman Universal PCR Master Mix, No Amp Erase UNG (Applied Biosystems, Foster City, CA, USA), and Taqman microRNA assay (Takara, Otsu, Shiga, Japan) for the miR-Let7A of interest. The PCR amplification was performed in ABI 7500 Real Time PCR (Life Technologies Corporation, CA, USA) at 95 • C for 10 min, followed by 40 cycles of 95 • C for 15 s and 60 • C for 60 s. The PCR incubation profile was extended to 40 cycles for miR-Let7A. The PCR primers for Let-7A were (F): 5 ′ -GCGCCTGAGGTAGTAGGTTG-3 ′ and (R): 5 ′ -CAGTGCAGGGTCCGAGGT-3 ′ . The PCR primers for U6 were (F): 5 ′ -CTCGCTTCGGCAGCACATATACT-3 ′ and (R): 5 ′ -ACGCTTCACGAATTTGCGTGTC-3 ′ , respectively. The data were uniformly normalized to the internal control U6, and relative expression levels were evaluated using the 2 − Ct method. All experiments were run in triplicate (Zhang et al., 2014).

Statistical Analysis
Statistical analyses were carried out using SPSS 18.0 software (IBM Corp., Armonk, NY, USA). All data are expressed as mean ± standard error of the mean (S.E.M.). Significant group differences were determined by one-way analysis of variance followed by Bonferroni post-hoc multiple comparisons tests. Each experiment consisted of three replicates per condition. Differences were considered statistically significant at * p < 0.05 and * * p < 0.001.

Inhibition of ASK1 Activation Enhances Cell Viability against High-glucose Stress
To investigate the viability of BV2 microglia in high-glucose conditions, we conducted an MTT assay with different concentration of glucose ( Figure 1A). Cell viability was reduced by glucose treatment (25, 60, 90, or 120 mM). After pretreatment with ASK1 inhibitor (600 nM, TOCRIS, Bristol, UK), the viability of high-glucose-exposed microglia significantly increased by 20% compared with that of the high-glucose treatment alone group ( Figure 1A). Because cell viability showed the largest change by ASK1 inactivation in the 120 mM glucose condition, we used this concentration in subsequent experiments. To determine the whether ASK1 directly participates in the alteration of cell viability against high-glucose stress, we conducted quantitative real time-PCR using ASK1-specific primer (Figures 1B,C). ASK1 expression was increased in the 25 mM glucose condition and was markedly reduced after ASK1 inhibitor pre-treatment ( Figure 1B). ASK1 expression was increased in the 120 mM glucose condition and was significantly reduced after ASK1 inhibitor pre-treatment ( Figure 1C).

Measurement of M1-and M2-type Microglia after High-glucose Injury
To observe the function of microglia, we investigated the expression of CD68 (Figure 2A) and CD206 ( Figure 2B) in 25 mM and 120 mM high glucose concentration using immunocytochemical analysis. The expression of CD68 was considerably increased in the high-glucose treatment group (120 mM) and was attenuated by the inhibition of ASK1 activation (Figure 2A). The expression of CD206 was considerably reduced in the high-glucose treatment group (120 mM) and was slightly increased by the inhibition of ASK1 ( Figure 2B).

Measurement of Cytokine Levels in Microglia after High-glucose Injury
To examine the mRNA level of cytokines in microglia following the inhibition of ASK1 activation in high-glucose injury (25 mM and 120 mM), we conducted quantitative real-time PCR The graph showed that BV2 microglia in the high glucose treatment groups (25, 60, 90, 120 mM) exhibited decrease of cell viability compared to the normal control group. BV2 microglia in all ASK1 inhibitor pre-treatment groups exhibited increase of cell viability after high glucose injury compared to the normal control group. Especially, the microglia in the glucose 120 mM with ASK1 inhibitor conditions showed markedly the increase of cell viability comparison with ASK1 inhibitor treatment group. Each experiment included the six repeats per condition. (B) The measurement of relative quantitative mRNA level. The graph showed the mRNA level of ASK1 in glucose condition. In high glucose 25 mM treatment group, the level of ASK1 mRNA increased evidently compared to the no treatment group. ASK1 inhibitor pre-treatment attenuated the expression of ASK1 in microglia in spite of 25 mM high glucose condition. GAPDH was used as a control. Each experiment included the three repeats per condition. Each experiment included the three repeats per condition. Data were expressed as mean ± S.E.M, and were analyzed statistically using One-Way ANOVA, followed by Bonferroni's post hoc. **p < 0.001. (C) The measurement of relative quantitative mRNA level. The graph showed the mRNA level of ASK1 in glucose condition. In high glucose 120 mM treatment group, the level of ASK1 mRNA increased evidently compared to the no treatment group. ASK1 inhibitor pre-treatment attenuated the expression of ASK1 in microglia in spite of 120 mM high glucose condition. GAPDH was used as a control. Each experiment included the three repeats per condition. Each experiment included the three repeats per condition. Data were expressed as mean ± S.E.M, and were analyzed statistically using One-Way ANOVA, followed by Bonferroni's post hoc. *p < 0.05, **p < 0.001. RQ, relative quantitative value of mRNA via GAPDH.
Frontiers in Cellular Neuroscience | www.frontiersin.org ( Figures 3A-D) and RT-PCR ( Figure 3E). The mRNA level of pro-inflammatory cytokine TNF-α in microglia was increased in the high-glucose treatment group (Figures 3A,C) and was dramatically reduced by ASK1 inhibition (Figures 3A,C). The mRNA level of pro-inflammatory cytokine IL-6 in microglia was significantly increased in the high-glucose treatment group (Figures 3B,D) and was decreased by ASK1 inhibition (Figure 3B). The mRNA level of anti-inflammatory cytokine IL-10 in microglia was significantly decreased in the high-glucose treatment group ( Figure 3E) and tended to increase after ASK1 inhibition ( Figure 3E). Taken together, these results indicate that the inhibition of ASK1 activation regulates the expression of cytokines.

Measurement of miR-Let7A Level in BV2 Microglia after High-glucose Injury
To examine levels of miR-Let7A in microglia following the inhibition of ASK1 activation in high-glucose injury (25 mM and 120 mM), we conducted a Taqman assay (Figure 4). The level of miR-Let7A in microglia was significantly increased The mRNA level of TNF-alpha was increased in the high glucose treatment group (25 mM) and reduced in ASK1 inhibitor pre-treated high glucose group. (B) The mRNA amount of IL-6 was increased in the high glucose treatment group (25 mM) and significantly decreased in ASK1 inhibitor pre-treated high glucose group. (C) The mRNA level of TNF-alpha was increased in the high glucose treatment group (120 mM) and reduced in ASK1 inhibitor pre-treated high glucose group. (D) The mRNA amount of IL-6 was increased in the high glucose treatment group (120 mM) and significantly decreased in ASK1 inhibitor pre-treated high glucose group (120 mM). (E) The mRNA level of IL-10 exhibited decrease of expression in the high glucose treatment group (120 mM) whereas it significantly increased in ASK1 inhibitor pre-treated high glucose group (120 mM). GAPDH was used as a control. Each experiment included the 3 repeats per condition. Data were expressed as mean ± S.E.M, and were analyzed statistically using One-Way ANOVA, followed by Bonferroni's post hoc. *p < 0.05, **p < 0.001. RQ, relative quantitative value of mRNA via GAPDH.
in the high-glucose treatment group [25 mM ( Figure 4A) and 120 mM ( Figure 4B)] and was reduced by ASK1 inhibition (Figure 4). The level of miR-Let7A in microglia in the highglucose treatment group was almost the same as that in microglia with overexpression of Let7A plus ASK1 inactivation in the high-glucose condition (Figure 4).

Relationship between ASK1 and miR-Let7A in High-glucose Injury
To confirm a relationship between miR-Let7A and ASK1 activation in microglia in high-glucose injury, we conducted quantitative real-time PCR ( Figure 5A) and RT PCR (Figures 5B,C) and western blot analysis (Figures 5D-F). The mRNA level of ASK1 in microglia was significantly increased in the high-glucose treatment group and was reduced in the miR-Let7A mimic transfection group ( Figure 5A). Also, the mRNA level of ASK1 was reduced by Let7A mimic transfection under glucose 120 mM treatment condition ( Figure 5A). The mRNA level of ASK1 in BV2 cells was reduced by Let7A mimic treatment (Figure 5B), and ASK1 inhibitor treatment ( Figure 5C) in comparison with the only glucose 120 mM treatment group (Figures 5B,C). The activation of ASK1 [the phosphorylation of ASK1(p-ASK1)/ASK1 protein level] was increased in glucose 120 mM treatment group, while the increased phosphorylation of ASK1 in glucose 120 mM treatment was attenuated by the inhibition of ASK1 ( Figure 5E). The activation of ASK1 was reduced in Let7A treatment group ( Figure 5F).

Effect of ASK1 Inhibition and miR-Let7A Overexpression on Cytokine Expression in High-glucose Injury
To examine changes in cytokine mRNA levels in microglia after ASK1 inactivation and miR-Let7A overexpression under highglucose injury, we conducted quantitative real-time PCR. The mRNA level of pro-inflammatory cytokine IL-6 in microglia was significantly increased in the high-glucose treatment group ( Figure 6A) and was also increased in the Let7A mimic transfection group. In addition, the expression of IL-6 was decreased in the ASK1 inactivation group and in the ASK1 inhibition plus Let7A overexpression group after high-glucose injury ( Figure 6A). The mRNA level of anti-inflammatory cytokine IL-10 in microglia was reduced in the high-glucose treatment group ( Figure 6B) and was increased in the miR-Let7A Under high glucose stress, ASK1 inhibitor inhibited the reduced level of miR-Let7A compared with the high glucose treatment group. The co-treatment with glucose 25 mM and miR-Let7A mimic showed higher level of miR-Le7A than high glucose treatment group. U6 was used as a control. Each experiment included the three repeats per condition. Data were expressed as mean ± S.E.M, and were analyzed statistically using One-Way ANOVA, followed by Bonferroni's post hoc. **p < 0.001. (B) In high glucose 120 mM treatment group, miR-Le7A level was considerably increased compared to the no treatment group. Under high glucose stress, ASK1 inhibitor inhibited the reduced level of miR-Let7A compared with the high glucose treatment group. The co-treatment with ASK1 inhibitor and miR-Let7A mimic showed lesser level of miR-Le7A than high glucose treatment group. U6 was used as a control. Each experiment included the 3 repeats per condition. Data were expressed as mean ± S.E.M, and were analyzed statistically using One-Way ANOVA, followed by Bonferroni's post hoc. **p < 0.001. RQ, relative quantitative value of mRNA via U6; ASK1 inhibitor, ASK1 inhibitor (600 nM) pre-treatment and high glucose injury group/The inhibition of ASK1 activation group; Let7A mimic, Let7A over expression group in high glucose condition; anti-Let7A, Let7A inhibition in high glucose condition; Glucose (25 mM), glucose 25 mM treatment; Glucose (120 mM), glucose 120 mM treatment. overexpression group. In addition, the expression of IL-10 was increased in the ASK1 inactivation group in the high-glucose condition compared with that in the high-glucose treatment alone group. In the inhibition of ASK1 plus Let7A overexpression in high-glucose injury group, the expression of IL-10 was slightly increased compared with that in the Let7A mimic transfection group ( Figure 6B).

Alteration of N-Myc and c-Myc Expression by ASK1 Inhibition and Let7A Overexpression in High-glucose Injury
To examine the regulation of N-Myc and c-Myc by ASK1 inhibition and miR-Le7A overexpression, we analyzed the mRNA level of these factors using RT-PCR (Figure 7). The mRNA level of N-Myc was increased in the high-glucose condition ( Figure 7A). The inhibition of ASK1 activation markedly decreased N-Myc expression, whereas miR-Let7A overexpression did not result in large changes in N-Myc mRNA levels in the high-glucose condition ( Figure 7A). Moreover, the inhibition of ASK1 in the Let7A mimic transfection condition decreased N-Myc mRNA level to almost the same value as that for ASK1 inhibition under the high-glucose condition ( Figure 7A). Based on our findings, we assume that miR-Let7A links ASK1 to N-Myc in the high-glucose condition. In addition, the mRNA level of c-Myc was significantly increased in the high-glucose condition ( Figure 7B). The inhibition of ASK1 activation decreased c-Myc expression, and miR-Let7A overexpression led to a slight decrease of c-Myc mRNA level under the high-glucose condition compared with that for the high-glucose treatment alone group ( Figure 7B).
Moreover, the inhibition of ASK1 plus Let7A overexpression increased c-Myc mRNA level in the high-glucose condition ( Figure 7B).

Discussion
In the CNS, elevated glucose levels lead to severe neuronal death and impaired cognition (Duarte et al., 2008;McCrimmon et al., 2012). In hyperglycemic conditions, ASK1 is activated, involving apoptotic gene expression (F. Wang et al., 2015). miRNAs are small, 19− to 23−nucleotide RNA molecules that play a role in regulating protein expression in cells (Cai et al., 2010;Feng et al., 2010;Lu et al., 2010). miRNAs have been implicated in various cellular processes such as proliferation and apoptosis (Bartel, 2004;Bentwich et al., 2005). In particular, previous studies suggest that miR-Let7A is associated with apoptotic signaling in high-glucose conditions (Urbich et al., 2008;Iliopoulos et al., 2009;Zhu et al., 2011;Perez et al., 2013). Our results confirm the involvement of ASK1 in microglia cell viability and activation in high-glucose conditions, consistent with findings that ASK1 is associated with high-glucose-induced endoplasmic reticulum stress and apoptosis (Jiang et al., 2015;Wang et al., 2015). In addition, our results regarding the M1type marker CD 68 (Lemstra et al., 2007;Wojtera et al., 2012) and M2-type marker CD 206 (Stein et al., 1992;Mulder et al., 2014) indicate that the inhibition of ASK1 may induce activation of M2-type microglia and affect signaling that suppresses the inflammation response, suggesting that M2-type macrophages protect cells from inflammation (Varin and Gordon, 2009;Colton and Wilcock, 2010;Cherry et al., 2014). In the present FIGURE 5 | The measurement of ASK1 on microglia in high glucose stress. (A) The mRNA level of ASK1 was evidently increased by glucose treatment. Under high glucose stress, ASK1 inhibitor and the miR-Let7A overexpression group inhibited the decreased level of miR-Let7A compared to the normal control group. GAPDH was used as a control. Each experiment included the three repeats per condition. Data were expressed as mean ± S.E.M, and were analyzed statistically using One-Way ANOVA, followed by Bonferroni's post hoc. *p < 0.05, **p < 0.001, (B) The overexpression of Let7A was significantly decreased the mRNA of ASK1 in glucose 120 mM treatment condition in comparison with the only glucose treatment group. GAPDH was used as a control. Each experiment included the three repeats per condition. Data were expressed as mean ± S.E.M, and were analyzed statistically using One-Way ANOVA, followed by Bonferroni's post hoc. *p < 0.05. (C) The inhibition of ASK1 was significantly attenuated the mRNA of ASK1 in glucose 120 mM treatment condition in comparison with the only glucose treatment group. GAPDH was used as a control. Each experiment included the three repeats per condition. Data were expressed as mean ± S.E.M, and were analyzed statistically using One-Way ANOVA, followed by Bonferroni's post hoc. *p < 0.05. (D) The phosphorylation of ASK1/ASK1 level was increased by glucose 120 mM treatment compared to the normal control group. Also the activation of ASK1 was reduced in the glucose with Let7A overexpression in comparison with the only glucose treatment group. ß-actin was used as a control. Each experiment included the three repeats per condition. Data were expressed as mean ± S.E.M, and were analyzed statistically using One-Way ANOVA, followed by Bonferroni's post hoc. *p < 0.05,**p < 0.001. (E) The activation of ASK1 was reduced by ASK1 inhibitor treatment under glucose 120 mM treatment in comparison with the ASK1 inhibitor treatment group. β-actin was used as a control. Each experiment included the three repeats per condition. Data were expressed as mean ± S.E.M, and were analyzed statistically using One-Way ANOVA, followed by Bonferroni's post hoc. *p < 0.05, **p < 0.001. (F) The activation of ASK1 was reduced by Let7A mimic treatment compared with the no treatment group. β-actin was used as a control. Each experiment included the three repeats per condition. Data were expressed as mean ± S.E.M, and were analyzed statistically using One-Way ANOVA, followed by Bonferroni's post hoc. *p < 0.05. ASK1 inhibitor, ASK1 inhibitor (600 nM) pre-treatment and high glucose injury group/The inhibition of ASK1 activation group; Let7A mimic, Let7A over expression group in high glucose condition; anti-Let7A, Let7A inhibition in high glucose condition; p-ASK1: the phosphorylation of ASK1. study, a high-glucose state led to increases in the levels of proinflammatory cytokines such as TNF-α and IL-6 (Varnum and Ikezu, 2012) and decreases in the levels of anti-inflammatory cytokine such as IL-10 (Ledeboer et al., 2000). Furthermore, we found that inhibition of ASK1 attenuated the release of pro-inflammatory cytokines and promoted the release of anti-inflammatory cytokine such as IL-10 in high-glucose stress.
Moreover, more miR-Let7A expression occurred in the ASK1 activation condition than in the normal condition. Based on our results, we assume that miR-Let7A may be linked to the activation of ASK1 and may be involved in the expression of cytokines in hyperglycemia. In the present study, the transfection of miR-Let7A and anti-miR-Let7A led to ASK1 activation and altered the expression of cytokines. In particular, miR-Let7A FIGURE 6 | The mRNA level of cytokines on microglia in high glucose stress. The mRNA levels of IL-6, IL-10 in ASK1 inhibition and miR-Let7A overexpression were measured using quantitative real time-PCR. (A) The mRNA level of IL-6 was increased in the high glucose treatment group and reduced in ASK1 inhibitor pre-treatment group. In high glucose condition, IL-6 mRNA level was attenuated as the inhibition of ASK1 activation. (B) The mRNA level of IL-10 was reduced in the high glucose treatment group and increased in ASK1 inhibitor pre-treatment group. In high glucose condition, IL-10 mRNA level was attenuated through the overexpression of miR-Let7A whereas the inhibition of ASK1 activation was promoted the expression of IL-10. GAPDH was used as a control. Each experiment included the three repeats per condition. Data were expressed as mean ± S.E.M, and were analyzed statistically using One-Way ANOVA, followed by Bonferroni's post hoc. *p < 0.05, **p < 0.001. RQ, relative quantitative value of mRNA. tended to reduce pro-inflammatory cytokines more than antiinflammatory cytokines. miR-Let7A regulated the expression of cytokines more in the ASK1 inhibitor co-treatment group than the miR-Let7A mimic transfection only group. Several studies demonstrate that anti-inflammatory cytokines are regulated through inhibition of ASK1 activation (Liu et al., 2012), and ASK1 is involved in the production of inflammatory cytokines in macrophages (Matsuzawa et al., 2005;Osaka et al., 2007;Yuk et al., 2009). Considering previous evidence, our results indicate that Let7A miRNA may strongly regulate the expression of inflammatory cytokines by controlling ASK1 activation. Specifically, our results show that miR-Let7A may affect the expression of anti-inflammatory cytokines including IL-10 by The mRNA level of c-Myc showed increase of expression in the high glucose treatment group whereas it significantly was attenuated in ASK1 inhibitor pre-treated high glucose group. In ASK1 inhibition with Let7A overexpression group, microglia was increased c-Myc mRNA level in high glucose condition than only high glucose treatment group. GAPDH was used as a control. Each experiment included the four repeats per condition. Data were expressed as mean ± S.E.M, and were analyzed statistically using One-Way ANOVA, followed by Bonferroni's post hoc. *p < 0.05, **p < 0.001. RQ, relative quantitative value of mRNA. ASK1 inhibitor, ASK1 inhibitor (600 nM) pre-treatment and high glucose injury group/The inhibition of ASK1 activation group; Let7A mimic. Let7A over expression group in high glucose condition.
regulating ASK1 activation under glucose-induced stress. IL-10 is an anti-inflammatory cytokine that inhibits the production of pro-inflammatory cytokines including TNF-α, IL-1, and IL-6 (Joyce et al., 1994;Joyce and Steer, 1996;Sawada et al., 1999) in response to oxidative stress (Ledeboer et al., 2000;Molina-Holgado et al., 2001;Szczepanik et al., 2001), and its expression is also correlated with protection against neuronal damage (de Waal Malefyt et al., 1991;Fiorentino et al., 1991;Froen et al., 2002). Also, gene delivery of either IL-10 in the rat hippocampus alleviates deficits in learning and memory function (Lynch et al., 2004;Nolan et al., 2005). Based on our results, the promotion of IL-10 by miR-Let7A transfection may suppress the inflammatory response and protect neurons via ASK1 activation in hyperglycemic conditions. The Myc protein, as an unstable nuclear phosphorylated protein, is involved in many cellular mechanisms (Hann and Eisenman, 1984;Ramsay et al., 1986). c-Myc induces apoptosis signaling with mitogenic signals (Prendergast, 1999). c-Myc protein is phosphorylated at both Ser-62 and Ser-71 by JNK (Noguchi et al., 1999) and is linked to ASK1 signaling (Noguchi et al., 2001). c-Myc triggers the activation of ASK1 and the mitochondrial apoptosis pathway (Evan and Littlewood, 1998;Noguchi et al., 1999;Desbiens et al., 2003) and induces cell proliferation (Sears et al., 1999). Furthermore, c-Myc is a crucial factor in both ribosome biogenesis and p53 stabilization (Menne et al., 2007;Dai and Lu, 2008;Kim et al., 2011). A recent study demonstrates that the inhibition of p38 and ASK1 reduces N-myc transcript levels (Guldal et al., 2012). N-myc triggers the apoptosis of neuroblastoma in hypoxia stress (Rossler et al., 2001). In hepatocytes, N-myc also leads to apoptosis following serum deprivation (Ueda and Ganem, 1996). N-myc was originally identified by oncogene expression profiling of human neuroblastoma cells (Schwab et al., 1983). The elevated expression of N-myc increases the susceptibility of neuroblastoma cells to diverse types of cell death signaling (Schweigerer et al., 1990;Malynn et al., 2000;Rossler et al., 2001). In addition, N-myc plays a crucial role in microglia function by regulating inflammatory mediators (Jung et al., 2005). Several researchers have discovered that the N-myc downstream-regulated gene 2 (Ndrg2) plays a key role in reactive astrogliosis involving IL-6/STAT3 signaling (Takarada-Iemata et al., 2014), and N-myc controls cytokineinduced regulator IRE1 protein and c-Jun N-terminal kinase in pancreatic beta cells (Brozzi et al., 2014). Considering our results, we speculate that miR-Let7A regulates the expression of c-Myc and N-myc by involving ASK1 activation in highglucose conditions and that miR-Let7A may also be associated with the cytokine secretion pathway by regulating c-Myc and N-Myc protein. In conclusion, we suggest that miR-Let7A may affect microglia by involving ASK1 activation in hyperglycemia. The results of the present study increase our understanding of the relationship between miR-Let7A and ASK1 and the function of microglia in hyperglycemia-induced oxidative stress.

Author Contributions
JS performed the experiences and drafted the manuscript. JL discussed the findings and helped draft the manuscript.