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Sevoflurane-Induced miR-211-5p Promotes Neuronal Apoptosis by Inhibiting Efemp2

Sevoflurane exposure can result in serious neurological side effects including neuronal apoptosis and cognitive impairment. Although the microRNA miR-211-5p is profoundly upregulated following sevoflurane exposure in neonatal rodent models, the impact of miR-211-5p on neuronal apoptosis and cognitive impairment postsevoflurane exposure has not yet been elucidated. Here, we found that sevoflurane upregulated miR-211-5p and downregulated EGF-Containing Fibulin Extracellular Matrix Protein 2 (Efemp2, Fibulin-4) levels in vitro and in vivo. Sevoflurane's effect on miR-211-5p expression was based on enhancing primary miR-211 transcription. miR-211-5p targets Efemp2's mRNA 3′-untranslated region, reducing Efemp2 expression. RNA immunoprecipitation revealed significant enrichment of the miR-211-5p:Efemp2 mRNA dyad in the RNA-induced silencing complex. miR-211-5p mimics downregulated Efemp2, leading to phosphorylation of Smad2 and Smad3, upregulation of pro-apoptotic Bim, and mitochondrial release of allograft inflammatory factor 1 and cytochrome C. In contrast, miR-211-5p hairpin inhibitor (AntimiR-211-5p) negatively regulated this apoptotic pathway and reduced neuronal apoptosis in an Efemp2-dependent manner. Sevoflurane-exposed mice administered AntimiR-211-5p displayed reduced cortical apoptosis levels and near-term cognitive impairment. In conclusion, sevoflurane-induced miR-211-5p promotes neuronal apoptosis via Efemp2 inhibition. Summary statement: This study revealed the significance of sevoflurane-induced increases in miR-211-5p on the promotion of neuronal apoptosis via inhibition of Efemp2 and its downstream targets.


Introduction
Sevoflurane is regularly employed as an inhaled pediatric and adult anesthetic in the clinic due to its quick induction and recovery time and low blood/gas partition coefficient (Brioni et al., 2017). However, two prospective randomized parallel-group clinical trials have shown that sevoflurane exposure can advance postoperative cognitive dysfunction in adults (Liu et al., 2013a;Qiao et al., 2015). In pediatric patients, brain structure and neurocognitive development have been shown to be directly altered by sevoflurane, resulting in an elevated possibility of cognitive dysfunction (Backeljauw et al., 2015;Sun 2010;Vutskits & Xie, 2016). Moreover, previous studies in neonatal and adult animal models have linked sevoflurane inhalation to enhanced neuronal apoptosis (Chen et al., 2013;Liu et al., 2013b;Qiu et al., 2015;Zheng et al., 2013) and cognitive deficits (Jiang et al., 2017;Liu et al., 2013a;Satomoto et al., 2009;Takaenoki et al., 2014;Tao et al., 2014;Yu et al., 2016). Unfortunately, the mechanisms underlying sevoflurane-induced neurotoxicity and cognitive dysfunction have not been fully investigated.
MicroRNAs (miRNAs) are a class of small (20-23 nucleotides) noncoding RNAs which promote posttranslation suppression of gene expression. miRNAs attach to the 3 ′ -untranslated region (UTR) of mRNAs resulting in inhibition of their translation and/or their degradation. miRNAs have been linked to the regulation of apoptotic pathways in neuronal cells (Jimenez-Mateos & Henshall, 2013) and the development of central nervous system disorders Redell et al., 2009;Ziu et al., 2011). Although several miRNAs are profoundly upregulated following sevoflurane exposure in neonatal rodent models (e.g., miR-211, miR-632, miR-466b-1-3p, miR-196c-3p) (Fujimoto et al., 2015;Ye et al., 2016), the association between these key candidate miRNAs and the mechanisms behind sevoflurane-induced neurotoxicity and cognitive dysfunction have not been thoroughly investigated.
This study investigated whether apoptosis-related cell loss and cognitive deficits can be linked to sevoflurane-induced upregulation of the key candidate miRNA miR-211-5p. An increase in miR-211-5p expression was revealed in the brain during the 72-h period postsevoflurane exposure. In vivo and in vitro models were employed to show that miR-211-5p affects neuronal apoptosis postsevoflurane exposure via inhibition of EGF-Containing Fibulin Extracellular Matrix Protein 2 (Efemp2, Fibulin-4). Treatment with a miR-211-5p hairpin inhibitor (AntimiR-211-5p) reversed these negative effects.

Animals
This study was approved by the Ethics Committee of [hospital name] ([city name], [country]). The guidelines detailed in the Guide for the Care and Use of Laboratory Animals (NIH 85-23-2985) were adhered to during all animal procedures. The experimental procedures are fully detailed in the Supplementary Methods. PRISM version 6 was used to perform all statistical analyses (GraphPad Software, La Jolla, CA, USA). If the data was shown to have a normal distribution, either unpaired Student's t-testing, one-way analysis of variance (ANOVA) with Tukey post-hoc testing, or two-way ANOVA (condition [room air, sevoflurane, etc.] × treatment etc.]) with Tukey post-hoc testing were applied as indicated. The y-axis labels indicate the control groups used for each comparison.

Rapid Upregulation of Pro-Apoptotic miR-211-5p at the Transcriptional Level by Sevoflurane
To assess the effects of sevoflurane on miR-211-5p expression in vivo, we exposed mice to 5% sevoflurane or sham conditions (room air) mice for 4 h. As previously shown in neonatal rat brains (Fujimoto et al., 2015), miR-211-5p expression was significantly upregulated in the murine brain following sevoflurane exposure (Figure 1a). A rapid increase in miR-211-5p was observed as early as 1 h postsevoflurane exposure; this increase peaked at 6 h and was maintained at 72 h postsevoflurane exposure (Figure 1a). To assess the effects of sevoflurane on miR-211-5p expression in cortical neurons in vitro, we exposed primary rat cortical neurons (RCNs) to 3.4% sevoflurane or room air for three hours. Two in vitro models of cortical neuronal apoptosis, staurosporine and etoposide (Sabirzhanov et al., 2012), were employed as positive controls for apoptosis. RCNs were selected over murine cortical neurons due to their superior robustness under excitotoxic stress in vitro (Cunha-Oliveira et al., 2006;Nassogne et al., 1998;Yang et al., 2010;Yu et al., 1999). miR-211-5p levels were significantly increased at 3 h following sevoflurane exposure (Figure 1b). miR-211-5p levels were also significantly increased by 3 h after induction of apoptosis in both the etoposide and staurosporine models (Figure 1b).
The transcriptional inhibitor α-amanitin (α-Am) was used to investigate the rapid increase in miR-211-5p levels in response to sevoflurane exposure. Quantitative polymerase chain reaction (qPCR) was used to assess expression of primary miRNA-211 (pri-miR-211) and mature miR-211-5p in RCNs treated with room air or sevoflurane at 1, 3, and 6 h post-α-Am treatment (10 μg/mL). Inhibition of miR-211-5p transcription by α-Am under room air conditions caused rapid reductions in pri-miR-211 and mature miR-211-5p levels ( Figure 1c). pri-miR-211 and mature miR-211-5p levels rapidly increased following sevoflurane exposure ( Figure 1d). The addition of α-Am caused rapid reductions in pri-miR-211 and mature miR-211-5p levels, implying the role of enhanced transcription in sevoflurane's induction of miR-211-5p ( Figure 1e). A similar pattern of findings were found in the positive control etoposide model (Figure 1c to e). These results reveal that sevoflurane's effect on miR-211-5p expression is based on enhancing pri-miR-211 transcription.
The regulation of RCN apoptosis by miR-211-5p was assessed with miR-211-5p mimics. Apoptosis, as measured by lactate dehydrogenase (LDH) release and cleaved caspase-3 (CC-3) activity, was increased by miR-211-5p mimics in a dosage-dependent manner (Figure 1f and g). In order to assess whether miR-211-5p inhibition would impact sevoflurane-induced apoptosis, RCNs were transfected with a miR-211-5p hairpin inhibitor or control hairpin inhibitor (AntimiR-211-5p or AntimiR-Ctrl, respectively) and exposed to sevoflurane. AntimiR-211-5p significantly decreased sevoflurane-induced LDH release and CC-3 activity in a dosage-dependent manner (Figure 1h and i). A similar pattern of findings were found in the positive control etoposide and staurosporine models (Figure 1f to i).
RNA-induced silencing complex (RISC) is formed by binding of Argonaute 2 (Ago2) proteins to one strand of mature miRNA, which is used as a template in order to recognize and subsequently cleave complementary mRNA (Becker et al., 2019). Therefore, miRNA:mRNA target pairs, such as the miR-211-5p:Efemp2 dyad, can be confirmed by Ago2-associated RNA immunoprecipitation (RIP). RCNs were harvested 6 h postsevoflurane treatment (the time point with the highest miR-211-5p expression) and underwent RIP with Ago2 antibodies. Significant enrichment of Efemp2 and miR-211-5p mRNA in the RISC complex were revealed with qPCR after sevoflurane exposure (Figure 3b). A similar pattern of findings were found in the positive control etoposide model ( Figure 3b). These results show that sevoflurane enhances the RISC-based interaction of miR-211-5p and Efemp2 mRNA.
In vivo, qPCR revealed a decrease in cortical Efemp2 expression as early as 1 h postsevoflurane exposure; this decrease peaked at 24 h and was maintained at 72 h postsevoflurane exposure (Figure 3c). In vitro, qPCR analysis in RCNs showed a similar decrease in Efemp2 expression postsevoflurane exposure (Figure 3d). A similar pattern of findings were found in the positive control etoposide model ( Figure 3d).
As Efemp2 encodes the ECM protein Fibulin-4, a negative regulator of Tgf-β/Smad signaling in murine neurons (Hanada et al., 2007;McLaughlin et al., 2006) and human fibroblasts (Renard et al., 2010;Urban & Davis, 2014), the role of miR-211-5p in the Efemp2/Tgf-β/Smad signaling pathway was assessed in RCNs postsevoflurane exposure. Expression of Efemp2 was decreased, while Smad2 and Smad3 phosphorylation were increased, after sevoflurane exposure (Figure 3e to h). These sevoflurane-induced changes were rescued by AntimiR-211-5p (Figure 3e to h). Expression of pro-apoptotic AIF-1 in nuclear fractions was enhanced after sevoflurane exposure, which was abrogated by AntimiR-211-5p (Figure 3i and j). A similar pattern of findings were found in the positive control etoposide model (Figure 3e to j).

The Neuroprotective Effects of AntimiR-211-5p are Mediated by Efemp2
Small-interfering RNA (siRNA)-based Efemp2 knockdown was used to investigate the role of Efemp2 in AntimiR-211-5p's neuroprotective effects. Three siRNAs against Efemp2 were tested, and the most potent siRNA was selected for further experimentation (Supplemental Figure S2a). Sevoflurane induced LDH release from RCNs; this was not affected by the addition of Efemp2-specific small-interfering RNA (siEfemp2) (Supplemental Figure S2b). AntimiR-211-5p decreased sevoflurane-induced LDH release; notably, this effect was nullified by the addition of siEfemp2 (Supplemental Figure S2c). A similar pattern of findings were found in the positive control etoposide model (Supplemental Figure S2a to c).
The role of Efemp2 in AntimiR-211-5p's regulation of the Efemp2/Tgf-β/Smad signaling pathway was assessed in RCNs post-sevoflurane exposure. AntimiR-211-5p rescued sevoflurane-induced downregulation of Efemp2 and upregulation of Smad2 and Smad3 phosphorylation (Supplemental Figure S2d and e). These AntimiR-211-5p-induced changes were nullified by the addition of siEfemp2 (Supplemental Figure S2d and e). A similar pattern of findings were found in the positive control etoposide model (Supplemental Figure S2d and f).
AntimiR-211-5p Upregulates Efemp2 Expression, Downregulates Smad2/3 Signaling, and Reduces Apoptosis in Sevoflurane-Exposed Mouse Cortices Vehicle (PBS), AntimiR-Ctrl, or AntimiR-211-5p was administered to room air-exposed or sevoflurane-exposed mice by i.c.v. injection 15 min postprocedure. Western blot analysis was then used to assess Efemp2 expression and Smad2/3 phosphorylation 24 h postroom air or sevoflurane exposure. Levels of Efemp2 were significantly decreased, and phosphorylated Smad2 and Smad3 were significantly increased, following sevoflurane exposure (Figure 4a and b). These results suggest that the regulation of Smad2/3 postsevoflurane exposure is via Tgf-β1-dependent phosphorylation rather than changes in the expression of Smad2/3. Treatment with AntimiR-211-5p rescued these changes in Efemp2 expression and Smad2/3 phosphorylation (Figure 4a and b).

AntimiR-211-5p Rectifies Near-Term Motor Function and Cognitive Deficits Induced by Sevoflurane Exposure
Vehicle (PBS), AntimiR-Ctrl, or AntimiR-211-5p was administered to room air-exposed or sevoflurane-exposed mice by i.c.v. injection 15 min postprocedure. Then, several tests for motor and cognitive function were administered. The beam walk test was used immediately prior to sevoflurane exposure (day 0) and on days 1, 7, 14, 21, and 28 postexposure in order to investigate motor function. There were significant differences between the sevoflurane-exposed AntimiR-Ctrl and sham groups solely on days 1 and 7 postsevoflurane exposure (Figure 5a), indicating that sevoflurane adversely affects nearterm motor function. Of note, AntimiR-211-5p therapy rescued this effect in sevoflurane-exposed mice.
In order to assess spatial working memory, the Y-maze spontaneous alternation test was used immediately prior to sevoflurane exposure (day 0) and on days 1, 7, 14, 21, and 28 postexposure. A functional working memory was found in the sham mouse group on day 1 postexposure (81.4 ± 3.3% spontaneous alternation) (Figure 5b). However, spontaneous alternation was significantly decreased solely on day 1 postexposure in sevoflurane-exposed AntimiR-Ctrl mice (64.9 ± 2.0%). Of note, AntimiR-211-5p therapy rescued this effect in sevoflurane-exposed mice. In order to further assess spatial working memory, the Morris water maze (MWM) test was used immediately prior to sevoflurane exposure (day 0) and on days 1-5 (first trial) and days 10-14 (second trial) post-exposure. Over days 1-4 (first trial), there were significant increases in latency time required to find the hidden platform in sevoflurane-exposed AntimiR-Ctrl compared to sham mice, indicating that sevoflurane adversely affects spatial memory (Figure 5c). Notably, sevoflurane-exposed mice treated with AntimiR-211-5p partially rescued this increase in latency time. Similarly, during the first trial's probe test on day 5, we observed significantly higher latency times (Figure 5d), significantly lower number of crossings (Figure 5e), and significantly lower times in the target quadrant (Figure 5f) in sevoflurane-exposed AntimiR-Ctrl compared to sham mice, effects partially rescued by AntimiR-211-5p (Figure 5d to f). However, these changes in sevoflurane-exposed mice were not observed during the second trial's probe test on day 14 (Figure 5d to f), suggesting that sevoflurane's observable effects are near-term.
The NOR test was then used to assess retention memory on days 2-3 postsevoflurane exposure (Figure 5g and h). An intact memory was revealed in sham mice, who spent 15 s with the novel object during the choice phase on day 3 postsevoflurane exposure (Figure 5h). Sevoflurane-exposed mice treated with AntimiR-Ctrl or AntimiR-211-5p spent a similar time with the novel object compared to the sham group (Figure 5h), suggesting that sevoflurane has no observable effects on retention memory.

Discussion
As miRNAs are able to target a number of mRNAs, they are able to exert a large degree of control over gene expression within the brain (Henshall, 2014;Ponomarev et al., 2013). This study aimed to investigate whether postsevoflurane neuronal apoptosis and cognitive deficits could be better controlled via modification of miR-211-5p expression. We found that both in vitro sevoflurane exposure resulted in a rapid increase in miR-211-5p expression, while AntimiR-211-5p reduced sevoflurane-induced neuronal apoptosis. AntimiR-211-5p also promoted a reduction in markers of caspase-independent apoptosis (AIF-1 nuclear translocation) (Susin et al., 1999) and -dependent apoptosis (PARP, CC-3, and α-fodrin cleavage) (Siman et al., 2004), mitochondrial permeabilization, and expression of Bim. In fact, treatment of neuronal cells with miR-211-5p mimics alone induces apoptosis, indicating that miR-211-5p can independently drive the neuronal cell death pathway. These in vitro findings were validated by two in vitro neuronal apoptosis models: the protein kinase inhibitor staurosporine and DNA-damaging agent etoposide, which respectively promote mitochondrial release of cytochrome C and AIF-1 via caspase-independent and -dependent mechanisms (Yakovlev et al., 2004). miR-211-5p has been previously shown to impair murine neuronal viability as well as neurite growth and branching by directly downregulating the pro-neurogenic Nua kinase Nuak1, thereby accelerating neuronal loss and cognitive dysfunction (Fan et al., 2016). However, pro-neurogenic Nuak1 expression is paradoxically upregulated in the murine hippocampus following sevoflurane exposure (Hayase et al., 2016), suggesting that miR-211-5p's negative regulation of Nuak1 does not play a significant role in sevoflurane-induced neuronal loss and cognitive dysfunction. On the other hand, Efemp2 (encoding the ECM protein Fibulin-4) has previously been identified as a target of miR-211-5p in chondrocytes (Liu & Luo, 2019). Although Efemp2 is overexpressed in glioma tumor cells and plays an anti-apoptotic role in glioma cells , Efemp2's regulatory role in injury-induced neuronal apoptosis has not been investigated. Here, we found a rapid reduction in Efemp2 expression following sevoflurane exposure, which was validated in our etoposide model. We also found that Efemp2 downregulation is associated with increased Smad2/3 phosphorylation, Bim upregulation, and nuclear translocation of AIF-1, leading to neuronal apoptosis (Cregan et al., 2004). Our findings are consistent with previous studies showing that Efemp2 knockdown promotes Smad2/3 phosphorylation (Hanada et al., 2007;McLaughlin et al., 2006) and that enhanced Smad2/3 phosphorylation promotes pro-apoptotic Bim expression (Ramesh et al., 2008;Thi et al., 2013).
Of note, significantly enriched levels of Efemp2 and miR-211-5p were revealed within the RISC complex, suggesting that posttranscriptional Efemp2 mRNA processing is miR-dependent. Efemp2 downregulation coupled with Figure 5. AntimiR-211-5p rectifies near-term motor function and cognitive deficits induced by sevoflurane exposure. (a) Beam walk analysis for fine motor coordination and balance and (b) Y-maze spontaneous alternation analysis for spatial working memory function on days 0 (pre-exposure), 1, 7, 14, 21, and 28 in RA-exposed or sevoflurane-exposed mice treated with AntimiR-Ctrl or AntimiR-211-5p (n = 9 mice per cohort). All mice had ≤10-foot faults prior to RA or sevoflurane exposure. (c-f) MWM analysis for spatial memory function (n = 9 mice per cohort). Quantification of (c) escapes latency during the first four days after sevoflurane exposure as well as (d) escape latency, (e) number of crossings, and (f) time spent in the target quadrant during probe test on the fifth day (first test) and fourteenth day (second test) post-RA or sevoflurane exposure. (g, h) NOR, (g) sample phase, and (h) choice phase assessments for memory retention on days 2-3 post-RA or sevoflurane exposure (n = 9 mice per cohort). Data shown as means ± SDs. *p < .05, **p < .01 (two-way ANOVA [condition × treatment] with Tukey post-hoc test). Abbreviations: AntimiR-211-5p = miR-211-5p hairpin inhibitor; RA = room air; MWM = Morris water maze; NOR = novel object recognition; ANOVA = analysis of variance. nuclear translocation of AIF-1 by AntimiR-211-5p indicates a key role for the miR-211-5p/Efemp2 axis in neuronal cell death. Furthermore, Efemp2 silencing was able to reverse AntimiR-211-5p-dependent neuroprotection. Mature miRNA catabolism is relatively variable with a quick turnover rate compared to its processing, which is constant (Krol et al., 2010). The rate of miR-211-5p catabolism in RCNs was shown to be fast, and the rate of decay was not affected by cellular injury. Exposure to sevoflurane (or etoposide) resulted in increased levels of α-Am-inhibitable pri-miR-211 and miR-211-5p expression, which suggests that a higher rate of pri-miR-211 transcription is the cause of sevoflurane-induced miR-211-5p upregulation. Further investigation is needed into the mechanism involved in the upregulated transcription of miR-211-5p, and possibly its host gene transient receptor potential cation channel subfamily M member 1 (Levy et al., 2010), following sevoflurane exposure.
We also found that in vivo sevoflurane exposure resulted in a rapid increase in miR-211-5p expression. The role of miR-211-5p in the promotion of in vivo sevoflurane-induced neuronal apoptosis was indicated by the neuroprotective effects exerted with AntimiR-211-5p. This treatment resulted in a reduction in sevoflurane-dependent activation of Bim and other downstream targets in the injured murine cortex. This was associated with a decrease in sevoflurane-induced α-fodrin cleavage, mitochondrial release of cytochrome C and AIF-1, CC-3 expression, and cortical neuronal death. Furthermore, we revealed an amelioration of near-term motor and cognitive functional deficits with AntimiR-211-5p therapy. Significant improvements in motor and cognitive parameters after treatment with the AntimiR-211-5p were found on days 1-7 postsevoflurane exposure.
There have been limited reports on the impact of sevoflurane anesthesia on levels of Efemp2 expression. Profound downregulation of Efemp2 has been reported by one murine study (Hayase et al., 2016), whereas one rat study reported no significant changes in Efemp2 expression (Pan et al., 2011). However, these previous studies performed no further experiments on Efemp2's role in sevoflurane exposure. This study is the first to reveal that sevoflurane exposure results in the reduction of Efemp2 mRNA and protein expression in the injured murine cortex and that neuronal Efemp2 may provide neuroprotection from sevoflurane exposure.
In conclusion, this study revealed the significance of sevoflurane-induced increases in miR-211-5p on the promotion of neuronal apoptosis via inhibition of Efemp2 and its downstream targets.

Ethical Approval
This study was approved by the Ethics Committee of Jiangxi Provincial People's Hospital Affiliated with Nanchang University (Nanchang, China). The Guide for the Care and Use of Laboratory Animals (NIH 85-23-2985) guidelines were adhered to during all animal procedures.