KCNQ1OT1 promotes autophagy by regulating miR‐200a/FOXO3/ATG7 pathway in cerebral ischemic stroke

Abstract Dysregulation of long noncoding RNAs (lncRNAs) is associated with abnormal development and pathophysiology in the brain. Increasing evidence has indicated that ischemic stroke is becoming the most common cerebral disease in aging populations. The treatment of ischemic stroke is challenging, due in part to ischemia and reperfusion (I/R) injury. In this study, we revealed that potassium voltage‐gated channel subfamily Q member 1 opposite strand 1 (KCNQ1OT1) was significantly upregulated in ischemic stroke. Knockdown of KCNQ1OT1 remarkably reduced the infarct volume and neurological impairments in transient middle cerebral artery occlusion (tMCAO) mice. Mechanistically, KCNQ1OT1 acted as a competing endogenous RNA of miR‐200a to regulate downstream forkhead box O3 (FOXO3) expression, which is a transcriptional regulator of ATG7. Knockdown of KCNQ1OT1 might inhibit I/R‐induced autophagy and increase cell viability via the miR‐200a/FOXO3/ATG7 pathway. This finding offers a potential novel strategy for ischemic stroke therapy.

2018). Autophagy is a necessary process to degrade impaired organelles and misfolded proteins in eukaryotic cells. It can affect cellular homeostasis during synthesis and catabolism (Kim et al., 2013). Activated autophagy has been detected in numerous diseases, including cancer, AIDs, cardiac-cerebral vascular and neurodegenerative disorders, and can be either beneficial or detrimental, as a double-edged sword (Abdellatif, Sedej, Carmona-Gutierrez, Madeo, & Kroemer, 2018;Grishchuk, Ginet, Truttmann, Clarke, & Puyal, 2011;Zhou & Spector, 2008). Reduction in autophagy has been demonstrated to ameliorate damage caused by focal cerebral infarction (Xing et al., 2012). Autophagy-related genes are conserved from yeast to mammals. ATG7 encodes an E1-like ligase, which is essential to form a mature autophagosomal membrane. ATG7 is involved in regulation of cell death (Pattison, Osinska, & Robbins, 2011). As a critical promoter of autophagy, ATG7 is associated with multiple diseases, including cancer, metabolic and neurological diseases (Hu et al., 2018;Martinez-Lopez & Singh, 2016;Xie et al., 2016). Specific knockout of ATG7 protects against brain injury following hypoxia (Xie et al., 2016). Thus, we are interested in the molecular regulation of ATG7-dependent autophagy in cerebral I/R injury.
MicroRNAs (miRNAs) are a set of endogenous short noncoding RNAs that can affect mRNA stability, transcription, and translation (Ameres & Zamore, 2013). MiRNAs function by partially or completely combining with the 3'-UTR of their targeted mRNAs . Accumulated evidence has shown that lncRNAs can serve as molecular sponges of miRNAs in binding to their downstream genes. For instance, lncRNA MIAT directly combines with miR-150-5p and linc00152 is directly targeted by miR-103a-3p in a specific-sequence manner (Yan et al., 2015;Yu et al., 2017).
Bioinformatics software (Lncbase) predicts a latent binding site for KCNQ1OT1 and miR-200a. And we realized that the increase in miR-200a was greater than others after KCNQ1OT1 knockdown in oxygen and glucose deprivation and re-oxygenation (OGD/R) cells.
MiR-200a was determined to participate in inflammatory and senescence processes in cells (Zhao et al., 2018). In addition, miR-200a inhibits proliferation in carcinoma cells . It was reported that miR-200a regulated autophagy in ovarian cancer via the ATG7 pathway (Hu et al., 2018). MiR-200a, which is upregulated by thymosin beta 4, could prevent cell death after ischemic injury by regulating the EGFR signaling pathway (Santra et al., 2016). However, the detailed function of miR-200a in I/R-induced brain damage needs further exploration.
Forkhead box O3 (FOXO3) was a speculated target of miR-200a in the miRWalk database. According to previous studies, FOXO3, which belongs to the FOXO family, is a transcription factor that regulates cell autophagy, survival, and senescence in mammals (Accili & Arden, 2004). FOXO3 induces autophagy and inhibits cell proliferation in gastric adenocarcinoma (Gao et al., 2018). A FOXO3-related pathway is involved in ketone neuroprotection against ischemic stroke (Yin, Han, Tang, Liu, & Shi, 2015). Evidence has shown that increased FOXO3, which is mediated by miRNA-132 and miRNA-212, was able to impair cell viability (Wong et al., 2013). Additionally, FOXO3 was identified to direct the autophagy program through regulating multiple downstream genes (Warr et al., 2013). Using the JASPAR database, we found that ATG7 was among the downstream targets of FOXO3. After we confirmed the effect of ATG7-dependent autophagy in I/R injury, we hypothesized that FOXO3 might function by targeting ATG7.
In the present study, we detected KCNQ1OT1,FOXO3, and ATG7 expression in tissues and cells after I/R injury. In addition, we explored possible mechanisms that may influence cell viability and autophagy in I/R. We explain the involvement of the KCNQ1OT1/miR-200a/FOXO3/ATG7 axis in regulating I/R-induced autophagy. These results may provide a novel understanding of molecular regulation in I/R injury and offer an alternative approach to improve ischemic stroke treatment.

| KCNQ1OT1 was upregulated in focal ischemia
To determine whether KCNQ1OT1 was related to ischemic stroke, we tested relative expression in AIS patients using Quantitative realtime PCR (qRT-PCR). The basic characteristics of participants are listed in Table 1. Compared with controls, KCNQ1OT1 was markedly increased in AIS patients' plasma ( Figure 1a). In addition, KCNQ1OT1 expression was positively correlated with stroke severity, which was evaluated based on National Institute of Health Stroke Scale (NIHSS) scores (R 2 = 0.2170, p < 0.01, Figure 1b). We established the transient middle cerebral artery occlusion (tMCAO) model to simulate I/R in vivo and detected KCNQ1OT1 expression in mice plasma and brain tissue (Figure 1c, d). Statistical analysis revealed that KCNQ1OT1 expression in plasma was positively correlated with expression in brain tissue in tMCAO mice (R 2 = 0.6342, p < 0.01, Figure 1e). Autophagy was activated in I/R injury, with increased ATG7 expression in mice subjected to tMCAO (Figure 1f).

| KCNQ1OT1 knockdown alleviated brain injury and inhibited autophagy in tMCAO mice
To investigate the role of KCNQ1OT1 in ischemic stroke in vivo, mice were preinjected with either sh-KCNQ1OT1 lentivirus or negative control (sh-NC) lentivirus into their lateral ventricles two weeks before the tMCAO operation. Mice were then sacrificed 24 hr after tMCAO (Supporting Information Figure S1a). Markedly downregulated KCNQ1OT1 expression was observed in the sh-KCNQ1OT1-injected mice compared with the sh-NC-injected group (Supporting Information Figure S1b). Cerebral infarction volume in mice was stained using 2,3,5-triphenyltetrazolium chloride (TTC) ( Figure 1g). The infarct volume was significantly reduced in the mice injected with sh-KCNQ1OT1 compared to the sh-NC group ( Figure 1h). Autophagy was inhibited with lower ATG7 expression in tMCAO mice, which were pretreated with sh-KCNQ1OT1 lentivirus (Supporting Information Figure S1c). To explore the potential influence of KCNQ1OT1 on the development of ischemic stroke, we recorded neurological deficit scores at 1, 3, 7, and 14 days after tMCAO. The data indicated that KCNQ1OT1 knockdown ameliorated neurological impairments in tMCAO mice (Supporting Information Figure S1d). These results suggested that KCNQ1OT1  (c) and brain tissue (d) of tMCAO and sham mice detected by qRT-PCR. Data are presented as the mean ± SD (n = 10 in each group). ***p < 0.001 vs. sham group. (e) Linear regression analysis was conducted to each individual KCNQ1OT1 expression in plasma and brain tissue in tMCAO group (n = 10), **p < 0.01. (f) Protein levels of ATG7, SQSTM1, and LC3B II in tMCAO and sham mice with GAPDH as an endogenous control. Data are presented as the mean ± SD (n = 6 in each group). *p < 0.05 vs. sham group. (g) Infarct region was visualized by triphenyltetrazolium chloride (TTC) staining. (h) Infarct size was measured using Image J software. Data are presented as the mean ± SD (n = 6 in each group). *p < 0.05 vs. tMCAO + sh-NC group

| KCNQ1OT1 knockdown protected cells in OGD/R by inhibiting autophagy
Cells were exposed to OGD/R treatment to mimic I/R in vitro.
We investigated the regulating mechanism of KCNQ1OT1 in the OGD/R model. We first established the stable sh-KCNQ1OT1  3-methyladenine (3-MA) as an autophagy inhibitor or rapamycin to induce autophagy. As shown in Figure 2c, impairment of cell viability in OGD/R-treated cells was alleviated after KCNQ1OT1 knockdown or 3-MA intervention. This change generated after transfection of sh-KCNQ1OT1 could be abolished by rapamycin treatment. In addition, autophagy was inhibited in sh-KCNQ1OT1-transfected OGD/R cells, while inhibition could be rescued by rapamycin treatment (Figure 2d).
Since apoptosis was identified as crucial programmed cell death, we focused on the impacts of autophagy on OGD/R-induced apoptosis. We detected the cell apoptosis rate using flow cytometry analysis and terminal-deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay. The cell apoptosis rate was increased in OGD/R-treated cells and became lower after treatment with either sh-KCNQ1OT1 plasmids or 3-MA. Rapamycin intervention negated the reduction in apoptosis rates due to KCNQ1OT1 knockdown ( Figure 2e; Supporting Information Figure S2a).
To further explore the activation of autophagy, we immunostained the autophagosome marker LC3B and found that transfection with sh-KNQ1OT1 markedly suppressed OGD/R-induced autophagy (Supporting Information Figure S2b). In addition, autophagic vacuoles (AVs) were observed by transmission electron microscopy (TEM), which were characterized as sequestration of organelles or portions of the cytoplasm with double membranes (Klionsky et al., 2016). Counting showed that the increased number of AVs in OGD/R cells could be inhibited after transfection with sh-KCNQ1OT1  RFPs was observed in early autophagosomes, which appeared as yellow puncta. When autolysosomes were formed with the fusion of autophagosomes and lysosomes, pH value in cells fell below 5, and the GFPs were subsequently degraded with only the RFPs left . The fluorescent density and intensity of yellow and red puncta were greatly increased after OGD/R treatment, whereas fewer yellow and red puncta occurred in the sh-KCNQ1OT1-transfected group (Supporting Information Figure S2c).
These results suggested that activated autophagy could promote cell apoptosis in I/R injury. KCNQ1OT1 knockdown might protect cells by mediating I/R-induced autophagy.

| miR-200a promoted cell survival in OGD/R
To further profile the molecular mechanism of KCNQ1OT1 regulating autophagy in I/R injury, we explored miRNA expression to determine miR-200a as the most affected downstream molecular target using miRNA microarray (Supporting Information Figure S3a).
MiR-200a had been previously proven to be conducive to cell survival in ischemia (Santra et al., 2016). In this study, miR-200a was

| KCNQ1OT1 knockdown inhibited autophagy by upregulating miR-200a
Data indicated a negative correlation between KCNQ1OT1 and miR-200a expression in the brain tissue of tMCAO mice (R 2 = 0.6875, p < 0.01, Supporting Information Figure S3c). To assess KCNQ1OT1 regulation of miR-200a in I/R injury, we detected miR-200a expression in sh-KCNQ1OT1 cells following OGD/R and showed that miR-200a expression was increased in cells transfected with sh-KCNQ1OT1 (Supporting Information Figure S3d).

| FOXO3 was upregulated and induced autophagy in OGD/R
FOXO3 has been reported to be associated with ischemic stroke (Yin et al., 2015) and may participate in regulation of autophagy (Warr et al., 2013). To verify the role of FOXO3 in I/R injury, we detected FOXO3 protein levels both in brain tissues and cells. As shown in Figure 4a

| miR-200a targeted FOXO3 3'-UTR and inhibited its expression
Given that FOXO3 was confirmed as an autophagy motivator in I/R injury, we hypothesized that FOXO3 may be involved in autophagy modulated by KCNQ1OT1. First, we recognized that FOXO3 expression was decreased in OGD/R cells after KCNQ1OT1 knockdown

| ATG7 was involved in FOXO3-mediated autophagy in OGD/R
ATG7 is known as an E1-like ligase, which is essential in autophagy processes (Mizushima & Komatsu, 2011;Nishida et al., 2009;Pattison et al., 2011). Knockout of ATG7 could promote cell survival during hypoxia (Xie et al., 2016). ATG7 was predicted to be directly targeted by FOXO3 in the JASPAR database. The present study found a positive correlation between FOXO3 and ATG7 in the brain of mice subjected to tMCAO ( Figure 6a). As shown in Figure  regulation of ATG7, cell lines with stable transfection of ATG7 knockdown were established (Supporting Information Figure   S4a). As shown in Supporting Information Figure S4b To investigate whether FOXO3 might bind with the promoter of ATG7, luciferase assays were performed. The promoter sequence of ATG7 was determined by searching the Ensembl Genomes database. (c) Different reporter constructs with schematic depiction was applied, and the luciferase activity is detected. The Y-bar shows the deletions on the DNA fragments. X-bar shows the plasmid activity with normalized to the co-transfection of reference vector, and relative activity to pEX3 empty vector, which activity was set to 1. Data are presented as the mean ± SD (n = 3, each). (d) Presentation of the predicted binding site for FOXO3 and ATG7 promoter region 3,000 bp upstream of the transcription start site (TSS) which designated as +1. Immunoprecipitated DNA was amplified by PCR. Normal rabbit IgG was used as a negative control. (e) The schematic description of the mechanism of KCNQ1OT1/miR-200a/FOXO3/ATG7 axis in regulating autophagy during ischemia and reperfusion (I/R) injury ( Figure 6c). These results indicated that the necessary element affecting an increase in ATG7 promoter activity was likely located in the −425 site region.
To further verify whether FOXO3 directly targeted the promoter of ATG7, chromatin immunoprecipitation (ChIP) assays were conducted. For a negative control, PCR was applied to amplify the 2,000 bp upstream region of the putative FOXO3 binding site, which was not considered to be associated with FOXO3.
Immunoprecipitation results illustrate a direct association between FOXO3 and the ATG7 promoter at putative binding site 1 (Figure 6d).
However, there were no associations between FOXO3 and other putative ATG7 binding sites and control regions. This finding indicated that FOXO3 likely modulated ATG7 at the transcriptional level. likely hindered the neuroprotective effects of miR-200a by directly binding with its 3'-UTR. ATG7 was essential in autophagy activation.

| D ISCUSS I ON
Knockdown of ATG7 could negate the influence of FOXO3 on cell viability in OGD/R, and FOXO3 targeted ATG7 promoter directly.
Ischemic stroke is difficult to treat due to the complex processes involved in I/R injury, including apoptosis and autophagy. Autophagy has been proven to be a continuous physiological process and likely to aggravate apoptosis through excessive degradation (Mizushima, Yoshimori, & Levine, 2010 is involved in tumor, brain injury, cardiomyopathy, and diabetic nephropathy (Fang et al., 2015;Na et al., 2018;Osei et al., 2018;Wei et al., 2014). In addition, miR-200a ameliorates cell impairment through ischemic preconditioning (Lee et al., 2010). Upregulation of miR-200a mediates cell protection in cerebral ischemia (Santra et al., 2016). In this study, we found that miR- Mounting evidence has indicated that miRNAs could mediate downstream gene expression by binding to their 3'-UTR regions (Ameres & Zamore, 2013). The miRWalk database predicted that miR-200a might target FOXO3 3'-UTR. FOXO expression is progressively increased in the aging brain to prevent axonal degeneration (Hwang et al., 2018). FOXO3 was confirmed to promote autophagy via various pathways (Warr et al., 2013;Zhou et al., 2012 (Hu et al., 2018). Our finding suggests a possible novel mechanism of miR-200a in regulating I/R-induced autophagy.
Previous evidence has implied that FOXO3 is associated with multiple genes during autophagy as a transcription factor (Warr et al., 2013). We tested the expression of several FOXO3 targets in OGD/R cells to find that ATG7 and LC3B expressing changed most obviously (Supporting Information Figure S5). In addition, CHIP assay confirmed ATG7 as a direct downstream gene of FOXO3. It was reported that myocardial infarction could induce increased ATG7 expression . Consistent with this finding, the results of the present study indicate that ATG7 is upregulated in I/R injury of the brain. ATG7 exerted crucial effects on autophagy, such as conversion from LC3B I to LC3B II and autophagic vacuole transport in the cytoplasm (Nishida et al., 2009). Early research found that ATG7dependent autophagy exacerbated cortical neuron death (Grishchuk et al., 2011). In this study, we revealed that FOXO3 might aggravate autophagy in I/R injury by directly promoting ATG7 expression as a transcription factor. FOXO3 is also likely to regulate autophagy by targeting other molecules or through other pathways in I/R injury of brain, which will require more exploration in the future.
In conclusion, these results revealed KCNQ1OT1 upregulation in I/R injury of the brain and underlying molecular mechanisms. This study illustrated the involvement of the KCNQ1OT1/miR-200a/ FOXO3/ATG7 axis in regulating autophagy induced by I/R injury.
These findings might provide a novel strategy in the treatment of ischemic stroke.

| Animals
All experiments related to animals were approved by the Institutional Animal Care and Use Committee of China Medical University. This study was conducted in complete compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
We spared no effort to minimize the stress and pain of animals used in this trial. Male C57BL/6J mice weighing 22 to 25 g (8-10 weeks old) were provided by Beijing HFK Bioscience Cooperation, China.
Mice were maintained in the standard environment of 22°C and 70% humidity in a 12 hr light/dark cycle, with access to food and water ad libitum. Lentivirus encoding short-hairpin RNA targeting KCNQ1OT1 and its nontargeting sequence were used to infect mice brain tissue. Mice were divided into four groups (n = 10 per group): sham, tMCAO, tMCAO + sh-NC, and tMCAO + sh-KCNQ1OT1.

| Transient middle cerebral artery occlusion
A tMCAO mouse model was used to establish transient focal cerebral ischemia in vivo on the basis of previous studies. After fasting for eight hours, anesthesia was induced in an acrylic chamber of 3% isoflurane mixed with 70% nitrogen and 30% oxygen and maintained with 1.5% isoflurane through a facemask. The surgery was performed under a stereomicroscope. Left carotid arteries were exposed through a longitudinal median neck incision. The common carotid artery (CCA) was blocked temporarily using artery clamps, and the external carotid artery (ECA) was distally incised with 6-0 suture ligation. A 6-0 silicone rubber-coated nylon monofilament (Beijing Cinontech Co. Ltd, Beijing, China) was inserted into the ECA from the incision and advanced 9-10 mm via the internal carotid artery (ICA) until the origin of the middle cerebral artery (MCA). After 60 min of occlusion, the monofilament was withdrawn and the clamp on CCA was removed for reperfusion. Sham mice underwent an identical procedure without occlusion as controls. During the operation, rectal temperature was controlled at 37 ± 0.5°C using a heating pad.

| Infarct volume measurement
Mice were sacrificed at 24 hr after tMCAO. Brains were cut into 2-mm-thick slices and stained with 2% 2,3,5-triphenyltetrazolium Cells were grown in a humidified environment of 5% CO 2 at 37°C.

| Oxygen and glucose deprivation and re-oxygenation and drug intervention in cells
To mimic ischemic/reperfusion (I/R) in cells, an OGD/R model was

| Quantitative real-time PCR
Total RNA was extracted from plasma, brain tissue, and cells using TRIzol reagent (Life Technologies Corporation, Carlsbad, CA, USA).
RNA concentrations were detected, and the quality was determined at 260/280 nm absorbance using an Ultra-micro UV Spectrophotometer (N50 Touch; Implen, Germany). One Step SYBR PrimeScript RT-PCR Kit (Takara Bio, Inc., Japan) was applied to determine KQNQ1OT1 expression. TaqMan

| Western blot
Tissues and cells were kept in ice-cold RIPA buffer with protease Membranes were subsequently incubated with HRP-conjugated secondary antibodies for another 2 hr at room temperature. Immunoblots were visualized by an enhanced chemiluminescence detection kit (ECL kit; Millipore, Billerica, MA) under a chemiluminescence imaging analysis system (Amersham Imager 600, GE, CT, USA). Relative integrated density values were calculated using Image J software.

| Cell transfection
The following plasmids were synthesized: short-hairpin KCNQ1OT1

| Cell viability assay
Cell viability was detected by Cell Counting Kit-8 (CCK-8, Dojindo, Japan) assay. N2a cells were seeded in 96-well plates overnight at a density of 10 4 cells per well. After OGD/R treatment, culture medium was added with 10 μl CCK-8 solution in each well and incubated for 2 hr at 37°C. Absorbance was determined at 450 nm using a microplate reader (BioTek, Winooski, VT, USA).

| Immunofluorescence staining
Cells were fixed with cold methanol at −20°C for 15 min and washed three times in phosphate-buffered saline (PBS) for 5 min each. Cells were blocked in 5% normal goat serum for 1 hr at room temperature.

| Transmission electron microscopy
Cells were prefixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) overnight and postfixed in 1% osmium tetroxide for another 2 hr at 4°C. After dehydration, infiltration, and imbedding, the samples were sectioned for TEM observation (Hitachi, Japan).

| Autophagy flux detection
Adenovirus encoding mRFP-GFP-LC3 (Hanbio, Shanghai, China) was applied to infect cells. Cells were cultured for another 48 hr after infection and subjected to OGD/R. Cells were then fixed with 4% paraformaldehyde, and DAPI was used to show the nuclei. Imaging was performed by a laser scanning confocal microscope (Zeiss, Germany).

| Mice miRNA microarrays
Microarray analysis was performed by Kangchen Bio-tech (Shanghai, China) together with sample preparation and microarray hybridization.

| Luciferase reporter assays
The supposed binding sites of miR-200a with KCNQ1OT1 and the FOXO3 3′-UTR fragment were cloned by PCR and inserted into a pmirGLO Dual-luciferase miRNA Target Expression Vector (Promega, Madison, WI, USA) to create a luciferase reporter vector (KCNQ1OT1wt and FOXO3-wt) (GenePharma). Corresponding mutants (KCNQ1OT1-mut and FOXO3-mut) (GenePharma) were constructed by gene alteration of the supposed binding sites. N2a cells were transfected with pmirGLO vectors and miRNA plasmids by Lipofectamine 3,000. Relative luciferase activities were evaluated using a Dual-Luciferase Reporter Assay System (Promega) 48 hr after transfection.
Different promoter fragments of ATG7 were amplified from the genomic DNA, which was subcloned into a pGL3-Basic-Luciferase vector (Promega). pEX3 plasmids were constructed with full-length mouse FOXO3 (GenePharma). The luciferase assay was conducted according to previous descriptions. The firefly luciferase activity was normalized by renilla luciferase activity.

| Chromatin immunoprecipitation assay
ChIP assay was implemented using a Simple Chip Enzymatic Chromatin IP kit (Cell Signaling Technology) on the basis of manufacturer's protocols as previous described. Briefly, cells were suspended in 1% formaldehyde to cross-link proteins to DNA and sonicated to average DNA fragment size. Lysates (2%) were applied as an input control. The other lysates were incubated with normal rabbit IgG or FOXO3 antibodies (Abcam, UK) for immunoprecipitation. DNA fragments were amplified by PCR. Primer sequences used for PCR are listed in Supporting Information Table S1.

ACK N OWLED G M ENTS
This work is supported by grants from the National Natural Science

CO N FLI C T O F I NTE R E S T
None declared.

AUTH O R S' CO NTR I B UTI O N S
SJY performed experiments, analyzed data, and wrote the manuscript; MJY analyzed bioinformatics and prepared the figures; XH, LLW, and ZQB performed statistical analyses; JF designed the experimental study and revised the manuscript. All authors approved the final version of the manuscript.