Protective effect of DLX6-AS1 silencing against cerebral ischemia/reperfusion induced impairments

Silencing of DLX6-AS1 reduced acute injury of cerebral neurons

Mice were treated with cerebral ischemia (IS) / reperfusion (R) on day 7 after being injected icv with lentivirus DLX6-AS1-shRNA1, and then DLX6-AS1 expression was first measured in the ischemic cerebral hemisphere. As shown in Supplementary Figure 1A, DLX6-AS1 was upregulated after ischemia for 1 h (IS 1 h) and following reperfusion for 24 h (R 24 h) (1.72 ± 0.19 arbitrary units (a.u.)) compared with that of the sham group (1.04 ± 0.04 a.u., P < 0.05). To establish the actions of DLX6-AS1 after cerebral I/R, the expression of DLX6-AS1 of the mouse brain should been interrupted. There were three interrupted sequences were designed and transfected into N2a cells. The results showed that DLX6-AS1 shRNA1 played the best interrupted effect on DLX6-AS1 expression (Supplementary Figure 1B, the results of other two shRNA were not shown here). Therefore, DLX6-AS1 shRNA sequence was chose for the further experiment. The mouse was injected icv with LV-DLX6-AS1-shRNA or LV-NEG on day 7 before cerebral I/R. and then neurological functions were evaluated in Morris water maze and pole tests from day 22 through day 28 after cerebral I/R. In comparison to the LV-NEG group, DLX6-AS1 expression was successfully downregulated (0.29 ± 0.06 a.u., Supplementary Figure 1A) after icv injection with LV-DLX6-AS1-shRNA. Behavioral assessments were used to assess neurological functions and TTC staining to determine the volume of each lesion.

According to the results of mNSS, the scores of ischemia in the IS 1 h/R 24 h group (10.00 ± 0.68) were greater than in the sham animal group (0.67 ± 0.42, P < 0.001, Figure 1B). However, when the DLX6-AS1 level was reduced, the score of the LV-DLX6-AS1-shRNA+IS 1 h / R 24 h group was lower than the LV-NEG+IS 1 h / R 24 h group. Similar results were found using Clark scoring (Figure 1B). Following TTC staining, we found that there was a more significant brain infarct in the IS 1 h / R 24 h group in comparison to the sham group and that silencing of DLX6-AS1 expression reduced the infarct volume in the IS 1 h / R 24 h group (Figure 1C).

Figure 1. DLX6-AS1 silencing inhibited the acute injury of cerebral neurons induced by brain ischemia/reperfusion. (A) Flow chart of the study investigating DLX6-AS1 function. (B) Average neurological scores of the Clark and mNSS tests. (C) Representative images and (D) Statistical chart for TTC staining. Values represent the mean ± SEM (n = 6 for each group). ***P < 0.001 vs sham; +P < 0.05, ++P < 0.01 vs IS 1 h/R 24 h + LV-NEG.

Silencing of DLX6-AS1 ameliorated long-term neurological impairments induced by cerebral ischemia/reperfusion in C57/BL6 mice

To investigate further the long-term effects of DLX6-AS1 in cerebral I/R, the Morris water maze test was utilized to assess impairments in memory function and pole tests were employed to evaluate motor functions, while cresyl violet staining was used to investigate brain atrophy. The latencies of escape of all mice groups exhibited downward trends with experience. In addition, there were significant changes in escape latencies between the DLX6-AS1-down+IS 1 h/R 24 h and the IS 1 h/R 24 h groups during reperfusion from day 22 through day 26. However, the IS 1 h/R 24 h group at day 28 showed a marked retardation in their escape latencies compared to the sham group (Figure 2A). The movement trajectories of the mice in each group were also measured. The time spent in the second quadrant (Figure 2B) was shorter in the IS 1 h/R 24 h group at day 26 than in the other 3 groups (P < 0.05, Figure 2B), which perhaps indicated that cerebral I/R induced memory deficits had occurred.

Figure 2. DLX6-AS1 silencing ameliorated long-term neurological dysfunction after ischemia. (A) the escape latency, (B) representative image and statistical evaluation of the Morris water maze and the target quadrant time tests (C) the pole test, (D) photomicrograph (cresyl violet stain) and statistical chart showing the regions of the cortex, corpus callosum, hippocampus, and midbrain of the brain. Values represent mean ± SEM (n = 6 in each group). *P < 0.05, **P < 0.01, ***P < 0.001 vs sham; +P < 0.05, ++P < 0.01 vs + LV-NEG-shRNA.

The motor functions of the experimental mice were assessed using the pole test. The total time a mouse spent turning and moving to the bottom of the rod was greater in the IS/R group (Figure 2C, P < 0.001). In comparison solely with the IS/R group, silencing of DLX6-AS1 significantly reduced the total time in the IS/R plus LV-DLX6-AS1-shRNA group. To determine whether silencing of DLX6-AS1 could suppress regional brain atrophy, the volumes of atrophic regions were measured (percentage of contralateral hemisphere). Extreme alterations in the cerebral IS/R were readily found in the hippocampus and striatum compared to with the sham-operated group, and these changes were alleviated by treatment with LV-DLX6-AS1- shRNA (cc: 53.2% ± 2.1%, P < 0.05; hippocampus: 15.9% ± 2.4%, P < 0.01; midbrain: 12.6% ± 1.1%; Figure 2D).

Silencing of DLX6-AS1 inhibited neuronal apoptosis

To detect the actions of DLX6-AS1 on neuronal apoptosis triggered by cerebral I/R, TUNEL staining and the cleaved expression of caspase-3 protein in ischemic cerebral hemispheres were determined in vivo. In comparison with the sham-operated group of mice, the ratios of neuronal TUNEL-positive cells (44.7 ± 4.5%, P < 0.001) and cleaved caspase-3 (1.87 ± 0.18, P < 0.01) were significantly higher in the IS 1 h/R 24 h group (Figure 3A, 3B). However, the ratio of TUNEL-positive cells and cleaved caspase-3 protein in the LV-DLX6-AS1-shRNA+IS 1 h/R 24 h group was significantly lower in comparison with solely the IS 1 h/R 24 h group (Figure 3A, 3B). These data revealed that silencing of DLX6-AS1 inhibited neuronal apoptosis induced by brain ischemia/reperfusion.

Figure 3. DLX6-AS1 silencing inhibited neuronal apoptosis induced by brain ischemia/reperfusion. (A) Representative images and statistics of TUNEL staining of brain sections for assaying neuronal apoptosis. (B) Cleaved caspase-3 protein expression, measured by western blotting. Data are given as the mean ± SEM (n = 6 in each group). ***P < 0.001 vs sham; ⁺⁺P < 0.01, ⁺⁺⁺P <0.001 vs IS 1 h/R 24 h + LV-NEG-shRNA.

Silencing of DLX6-AS1 reduced apoptosis of N2a and SH-SY5Y cell lines induced by OGD/R

In order to establish further the effects of DLX6-AS1 in vitro, N2a and SH-SY5Y cells were treated with OGD/R. According to the RT-PCR results, DLX6-AS1 was upregulated time-dependently in both cell lines (Supplementary Figure 2A). Additionally, DLX6-AS1 was at its highest level (3.00 ± 0.36) after OGD 3 h and following reoxygenation at 24 h compared with the control group (untreated cells) (1.15 ± 0.24, P < 0.01, Supplementary Figure 2A). Additionally, upregulation of DLX6-AS1 expression occurred in a time-dependent fashion in SH-SY5Y cells (Supplementary Figure 2B). DLX6-AS1 expression was increased compared to the controls (1.08 ± 0.13, Supplementary Figure 2B) after OGD 3 h and following reoxygenation for 12 h (2.40 ± 0.04, P < 0.01) or 24 h (1.85 + 0.16, P < 0.05), in comparison to controls (1.08 ± 0.13, Supplementary Figure 2B).

To confirm the actions of DLX6-AS1 on neuronal apoptosis, N2a cells were transfected with DLX6-AS1-shRNA for 48 h and then exposed to OGD for 3 h; followed by 24 h re-oxygenation. Apoptotic cell numbers (32.48% ± 3.26%) and expression levels of cleaved caspase-3 (2.27 ± 0 .30) elicited by OGD/R were greater than for untreated control cells (7.64% ± 0.56%, 1.01 ± 0.02, Figure 4A, 4B). Downregulation of DLX6-AS1 levels with DLX6-AS1-shRNA significantly decreased cell apoptosis (18.48 ± 1.03) and cleaved caspase-3 expression (1.38 ± 0.10) induced by OGD/R (Figure 4A, 4B). Consistent with the N2a cells finding, transduction of SH-SY5Y cells with DLX6-AS1 siRNA (Supplementary Figure 2B) significantly reduced apoptotic cell numbers and decreased the expression of apoptotic proteins cleaved caspase-3 (Figure 4C, 4D) to inhibited OGD-R-induced apoptosis. The data indicated that silencing of DLX6-AS1 also inhibited neuronal apoptosis induced by OGD/R in vitro.

Figure 4. DLX6-AS1 was upregulated in an OGD/R model and DLX6-AS1 silencing reduced apoptosis of N2a and SH-SY5Y cells induced by OGD/R. (A) Representative images and statistics of TUNEL staining of N2a cells used to confirm apoptotic changes (100X). (B) Cleaved caspase-3 protein levels measured by western blotting. (C) Representative images and statistics of TUNEL staining of SH-SY5Y cells used to confirm apoptotic changes (100X). (D) Cleaved caspase-3 protein levels of SH-SY5Y measured by western blotting. Values represent mean ± SEM (n = 3 in each group). **P < 0.01, ***P < 0.001 vs Ctrl; +P < 0.05, ⁺⁺P < 0.01, ⁺⁺⁺P < 0.001 vs OGD 3 h/R 24 h + NEG.

miR-149-3p as a potential target for DLX6-AS1

In order to investigate the mechanism of DLX6-AS1 effects, the databases of pita, miRanda and RNAhybrid were interrogated to look for a predicted possible target miRNA that DLX6-AS1 might act on as a ceRNA. As shown in Figure 5A, 180 miRNAs in total were predicted in the 3 databases examined. Interestingly, miR-149-3p exhibited high scores (Table 1), and the scores of mmu-miR-149-3p and hsa-miR-149-3p were surprisingly similar (Table 1, Supplementary Figure 3A). Hence, we speculate that DLX6-AS1 acts as a ceRNA and competes with miR-149-3p. Therefore, we set out to detect the DLX6-AS1 sponging ability of miR-149-3p. By analyzing bioinformatics data, it was established that DLX6-AS1 has 3 sites that can bind to miR-149-3p. Therefore, wild-type and mutant forms of DLX6-AS1 were constructed. The miR-149-3p bound to all 3 sites of DLX6-AS1. Mutants of DLX6-AS1 separately abolished the binding effects of DLX6-AS1 on miR-149-3p, activity detected by luciferase assays (Figure 5B).

Figure 5. miR-149-3p may be a target of DLX6-AS1. (A) Predicted binding miRNAs of DLX6-AS1 derived from 3 databases. (B) Luciferase assay results of DLX-AS1 wild type (WT) and mutant (MUT) construct binding with mmu-miR-149-3p. (C) Effects of a miR-149-3p mimic on apoptosis induced by OGD/R, as detected by TUNEL staining. (D) Effects of a miR-149-3p mimic on caspase-3 expression induced by OGD/R in N2a cells detected by western blotting. (E) Effects of a miR-149-3p inhibitor on apoptosis induced by OGD/R, as detected by TUNEL staining. (F) Effects of a miR-149-3p inhibitor on caspase-3 expression induced by OGD/R in N2a cells detected by western blotting. Values represent the mean ± SEM (n = 3 in each group). *P < 0.05, ***P < 0.001 vs NEG, **P < 0.01 vs Ctrl; ⁺P < 0.05 vs OGD 3 h/R 24h + NEG-mimic, ⁺⁺⁺P < 0.001 vs OGD 3 h/R 24 h + NEG; ^#P < 0.05, ^##P < 0.01 vs OGD 3 h/R 24 h + DLX6-AS1-shRNA.

Furthermore, miR-149-3p expression levels were determined both in vivo and in vitro and it was demonstrated that miR-149-3p levels were abridged in mice brains treated with IS 1 h/R 24 h (Supplementary Figure 3B). In addition, the smallest concentrations of miR-149-3p were revealed 12 h after reperfusion of the in vitro N2a cells OGD/R test (0.51 ± 0.039, P < 0.01) compared to control mice (Supplementary Figure 3C), which indicated an opposite pattern to that of DLX6-AS1 expression. To investigate further the relationship between miR-149-3p and DLX6-AS1, we constructed miR-149-3p mimic and inhibitor sequences. After transfection with an miR-149-3p mimic for 48 h, miR-149-3p was successfully overexpressed (31.82 ± 4.24, P < 0.001, Supplementary Figure 3D).

The data from TUNEL staining and western blotting revealed that when miR-149-3p was overexpressed there was a significant reduction of apoptosis in OGD/R N2a cells (ratio of TUNEL-positive cells: 20.25% ± 1.57%, cleaved caspase-3: 1.68 ± 0.09) compared to NEG+OGD 3 h/R 24 h cells (ratio of TUNEL-positive cells: 39.99% ± 5.76%, cleaved caspase-3: 2.41 ± 0.04; Figure 5C, 5D). However, after transfection with an miR-149-3p inhibitor, the degree of cellular apoptosis (the ratio of TUNEL-positive cells: 11.63% ± 0.73%, cleaved caspase-3: 2.04 ± 0.20) was increased in comparison to the NEG group (i.e., the ratio of TUNEL-positive cells: 3.75% ± 0.513%, cleaved caspase-3: 0.97 ± 0.02, Figures 5C, 5D). As previously demonstrated, silencing of DLX6-AS1 reduced the degree of apoptosis of neurons elicited by OGD/R. It is noteworthy that in cells transfected with DLX6-AS1-shRNA and miR-149-3p inhibitors exposed to OGD/R (the ratio of TUNEL-positive cells: 31.21% ± 2.11%, cleaved caspase-3: 2.82 ± 0.37), apoptosis increased more than in the DLX6-AS1-shRNA+OGD 3-h/R 24-h group (ratio of TUNEL-positive cells: 19.17% ± 1.98%, P < 0.01; cleaved caspase-3: 1.94 ± 0.06, P < 0.05, Figure 5E, 5F). The data showed that a miR-149-3p inhibitor reversed the anti-apoptotic effect of DLX6-AS1-shRNA in cells treated with OGD/R, indicating that miR-149-3p may well be a target of DLX6-AS1.

The target of BOK

To explore the mechanism(s) of miR-149-3p actions in cerebral I/R, the target of miR-149-3p was predicted using TargetScanVert software. The results showed that a binding site for miR-149-3p was conserved in the 3’-UTR region of BOK in both Mus musculus and Homosapiens (Figure 6A). Alignment of the miR-149-3p sequence with BOK was performed to reveal that the BOK coding sequence may be a significant target for miR-149-3p. To verify BOK expression, we detected BOK protein expression both in vivo and in vitro. Interestingly, when compared to the sham or control groups, the expression of BOK protein was increased in the IS 1 h/R 24 h (1.82 ± 0.18, P < 0.001, Supplementary Figure 4A) and the OGD 3 h/R 24 h (1.63 ± 0.06, P < 0.01; Supplementary Figure 4B) groups.

Figure 6. BOK may be a target of miR-149-3p. (A) The predicted binding sites of miR-149-3p and BOK by TargetScanVert. (B) BOK expression in the brain I/R model treated by LV-DLX6-AS1. (C–E) BOK protein expression following treatment with a DLX6-AS1 shRNA, miR-149-3p mimic or miR-149-3p inhibitor in N2a cells. (F) Representative images and statistics of TUNEL staining of N2a cells used to confirm apoptotic changes (100X). (G) Cleaved caspase-3 protein levels measured by western blotting. Values represent mean ± SEM (n = 3 in each group). **P < 0.01 vs Ctrl, ***P < 0.001 vs Ctrl or Sham or NEG; ⁺P < 0.05 vs IS 1 h/R 24 h + LV-NEG or OGD 3 h/R 24 h + NEG,⁺⁺P < 0 .01 vs OGD 3 h/R 24 h + NEG-mimic or OGD 3 h/R 24 h + NEG-siRNA, ⁺⁺⁺P < 0.001 vs OGD 3 h/R 24 h + NEG-siRNA; ^#P < 0.05 vs OGD 3 h/R 24 h + NEG or OGD 3 h/R 24 h + DLX6-AS1-shRNA.

For the purpose of verifying the regulatory mechanism of DLX6-AS1 with miR-149-3p on BOK, BOK protein expression was detected after silencing of DLX6-AS1 and before IS/R and OGD/R treatments. BOK protein was upregulated in cells with OGD/R or in brain tissue with cerebral I/R compared to the sham group. However, the reduction of DLX6-AS1 expression in LV-DLX6-AS1-shRNA+IS 1 h /R 24 h induced downregulation of BOK protein expression compared with that in the LV-NEG+IS 1 h /R 24 h group (Figure 6B). Similar results were found in the OGD/R model when transfected with DLX6-AS1-shRNA (Figure 6C). However, cells transfected with a miR-149-3p inhibitor exhibited upregulation of BOK protein expression compared with the DLX6-AS1-shRNA + OGD/R group (1.95 ± 0.12 vs 1.54 ± 0.07, P < 0.05, Figure 6D). Furthermore, overexpression of miR-149-3p following miR-149-3p-mimic transfection under the condition of OGD/R treatment obviously reduced BOK protein expression (1.43 ± 0.06, P < 0.01; Figure 6E) compared to that in NEG+OGD 3 h /R 24 h cells (1.77 ± 0.06). The results showed that DLX6-AS1 silencing inhibited BOK expression both in vivo and in vitro, which was reversed by a miR-149-3p inhibitor.

Next we determined the effect of BOK on neuronal apoptosis. We knocked down the expression of BOK in N2a cells via siRNA transfection and detected the effects of BOK on the apoptosis of N2a cells. BOK expression was successfully decreased by shRNA-1 (Supplementary Figure 4C). As expected, knockdown of BOK expression significantly inhibited OGD/R induced the ratio of TUNEL-positive cells (Figure 6F) and cleaved caspase-3 expression (Figure 6G) compared with that NEG treatment group. This finding was confirmed that BOK could promote OGD/R induced apoptosis. Taken together, these data suggest that DLX6-AS1 upregulates BOK expression by blocking miR-149-3p in OGD/R treatment N2a cells.

Middle cerebral artery occlusion in mice

Adult male C57/BL6 mice (Certificate No: SCXK (Q) 2015-0018; weight range: 18–25 g; age: 2 months, n = 124) were purchased from the Hubei Provincial Center for Disease Control and Prevention. Female mice were not used to avoid any influences of sex steroids. C57/BL6 mice were housed in a 12-h light/ dark cycle in a climate-controlled room at the Experimental Animal Center of Wuhan University of Science and Technology. All mice were allowed free access to food and water before the procedure was performed under optimal conditions

(12/12 h light/dark cycle; humidity 60% ± 5%; temperature 22°C ± 3°C). Our institution’s local experimental ethics committee assessed and approved the proposed experiments and research protocols. Focal cerebral ischemia was induced by transient middle cerebral artery occlusion (tMCAO) for 60 min [22]. Briefly, mice were deeply anesthetized using a 1–2% isoflurane oxygen/nitrous oxide mixture in a ratio of 30% and 69% administered through a mask applied to the face; body temperature was maintained at 37 ± 0.3ºC with a small animal heating platform. The left common carotid as well as the external and internal carotid arteries were exposed and a silicone-coated 6-0 suture was routed from the stump of the external to the internal carotid artery until the lumen of the middle cerebral artery was reached. The distances from the bifurcation of the internal and external carotid artery to the middle cerebral artery was 10 ± 0.5 mm. Laser Doppler Flowmetry (Moor Instruments, UK) was used to establish that occlusion had been successfully achieved. The same procedure was used for sham-operated animals with the exception that the suture was routed along the internal carotid artery before being immediately withdrawn.

The sham group was comprised of 35 mice. In the LV-NEG–treated group, the mortality rate was 6.8% (3 of 44 mice) and the exclusion rate was 6.8% (3 of 44 mice). In the LV-DLX6-AS1–treated mice, the mortality rate was 6.8% (3 of 45 mice) and the exclusion rate was 4.4% (2 of 45 mice). Animals with hemorrhage of the middle cerebral artery or complications during surgery were excluded. Our institutional animal-care and use committee approved the research study.

Assessments of mice behavior

The assessments were conduced 24 h after reperfusion. The Clark scoring method and the modified neurological severity score (mNSS) were used for behavioral analyses.

Clark focal scales

A focal neurological scale (Clark focal test) was used for detailed evaluation of neurological defects, which displays a high correlation with the underlying infarct volume (Clark et al., 1997). The Clark focal test addresses body symmetry, gait, climbing, circling behavior, front limb symmetry, compulsory circling and whisker responses of mice. For each of the 7 categories assessed, the items were summed to provide a total focal score that ranged from 0 to 28.

Modified neurological severity score (mNSS)

In the mNSS-points test, overall neurological function was evaluated following the guidelines of Chen et al [23]. The tests included the evaluation of motor responses (raising the mouse by the tail, placing the mouse on the floor), sensory (placing and proprioceptive tests), reflexive (reflex absence and abnormal movements) and balance (beam balance tests) deficits on a scale of 0 to 18 (0: normal score; 18: maximal deficit score).

Morris water maze

The water maze task consisted of a circular water tank (120 cm in diameter and 60 cm in height) filled with opaque water (21–23°C), and a round platform (6 cm diameter), which was submerged 1 cm beneath the surface of the water at the center of the second quadrant. Before the start of hidden-platform training, mice were allowed to acclimate to the testing environment for 30 min. Hidden-platform training was carried out within 90 s over 5 consecutive days (6 sessions), with 4 trials in each session. If the mice failed to find the invisible platform within 60 s, they were guided to the platform and allowed to stand on it for 15 s. We then recorded escape latencies to find the hidden platform, swimming paths and swimming velocities. After 5 consecutive days of hidden-platform training, mice were allowed to rest for 1 day, and on day 6 a probe test was conducted. The platform was removed and the mice were allowed to search the pool for 60 s. The time spent in each quadrant was then analyzed. Data were traced through a TM-Vision video-tracking system (Chengdu Taimeng Software Co. Ltd, Chengdu, Sichuan Province, China).

Pole test

As previously described [24], a pole test was conducted on the eighteenth experimental day and utilized to detect bradykinesia and/or the coordination of motor activity of the mice. Briefly, mice were positioned face upwards at the top of a wooden pole (1 cm diameter, 50 cm in length) and the time taken for a mouse to descend to the base recorded; the maximum permitted duration for this activity was 120 s. A total of 3 trials were conduced for each mouse and the median of the data across the trials calculated. Individual animals were ‘trained’ on 3 consecutive days before the experiment proper was conducted.

Intracerebroventricular (icv) injection

C57/BL6 mice were anesthetized with 10% chloral hydrate and appropriately positioned on a stereotactic frame. LV-DLX6-AS1-down (LV-DLX6-AS1-shRNA1) and LV-negative-EGFP (LV-NEG) mice were given icv injections into the right lateral ventricle (10 μL syringe, rate 1 μL/min in a 4 μL total volume (Table 2), and were maintained in position for 5 min after the injection. The infusion coordinates were -1.0 mm lateral, -2.5 mm dorsal/ventral and -0.22 mm anterior/posterior to the bregma, respectively. After recovery from surgery mice were returned to their housing until the experimental endpoint was reached.

2, 3, 5-triphenyltetrazolium chloride (TTC) staining

Twenty-four hours after reperfusion each mouse was anesthetized, the brain quickly removed and sliced into 2 mm sections the coronal direction. Each section was incubated in 2% TTC for 30 min at 37°C (zero light) before being fixed in paraformaldehyde (4% solution). The ischemic region (pale) of each section was determined using Image J software (ver. 1.61) and the volume of the brain infarct evaluated thus: volume / total volume × 100% [10].

Cresyl violet staining

Brain sections were post-fixed for 24 h at 4°C, and then immersed for 72 h in sucrose solution (30%, 4°C) to enable cryo-protection of the sections. To evaluate the degree of brain injury or atrophy, 20 μm specimen sections were stained with 0.1% cresyl violet. Next the ipsilateral and contralateral regional areas (hippocampus, striatum, corpus callosum, cortex, and midbrain) were examined (IMT i-Solution Inc, Canada).

TUNEL labeling

Sections of brain and cultured cells were labeled with the TUNEL Bright Red Apoptosis Detection Kit System (Vazyme Biotech, US). Specimens were viewed on a CX31-32RFL confocal microscope. Cells undergoing apoptosis and the total number of cells were measured (Image J software). The apoptotic ratio was : apoptotic cells / total cells × 100%.

Real-time RT-PCR

Total RNA was extracted with Trizol reagent (Invitrogen) according to the manufacturer’s instructions. The reverse transcription reaction was performed with 2 μg of total RNA using a First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, K1622, MA, US). For PCR amplification, cDNA was amplified using SYBR Green Real-time PCR Master Mix (Takara, Shiga, Japan) and 0.4 μM of each primer pair. Amplification was carried out starting with an initial step of 5 min at 95°C, followed by 40 cycles of the amplification step (95°C for 10 s, 60°C for 30 s, and 70°C for 10 s) for mouse DLX6-AS1, human DLX6-AS1, mouse β-actin, human β-actin, miR-149-3p and U6 (the primers sequences are shown in Table 2). All amplification reactions for each specimen were carried out in triplicate and the means of the threshold cycles were used to interpolate curves using the Bio-Rad CFX manager (Bio-Rad, Richmond, CA, US). Results are expressed as the ratio of DLX6-AS1 to β-actin and miR-149-3p to U6 and the value of the control group was set to 1.

Oxygen glucose deprivation/reperfusion model

SH-SY5Y and N2a cells were grown in DMEM high glucose solution with 1% penicillin-streptomycin and 10% fetal bovine serum in a humidified culture chamber (37°C, 5% CO₂ atmosphere). Cells were seeded in 6-well plates (3 × 10⁵ cells per plate) and 24 h later the medium was replenished with serum-free glucose-free medium. Next, to induce oxygen glucose deprivation, plates were exposed to an atmosphere of 95% N₂ and 5% CO₂ at 37°C in an experimental chamber. Following 3 h oxygen glucose deprivation (OGD), normal medium was added to the plates. Cells were cultured under normoxic conditions for 0, 6, 12, 24 and 48 h at 37°C in a 5% CO₂ atmosphere; control cells did not undergo OGD treatment.

Cell transfection

For 24 h before OGD, cells were placed (3 × 10⁵ cells) in 6-well plates and transfected with DLX6-AS1-shRNA1, DLX6-AS1-shRNA2, DLX6-AS1-shRNA3 (see Table 2), an miR-149-3p inhibitor, an miR-149-3p mimic or a negative control by Lipofectamine 2000 (Invitrogen, US). After we detected the inhibitory effect of DLX6-AS1-shRNA, shRNA1 was chosen for the next experiments and construction of LV-DLX6-AS1 (vide supra). After 48 h, cells were harvested for RT-PCR and western blotting or were treated with OGD/R.

Western blotting

The expression of caspase-3 and BOK proteins that had been cleaved were detected using western blotting. Brain tissues and cell specimens were extracted using a lysis buffer (RIPA, Applygen, Beijing) that had been supplemented with 0.1 mM phenylmethylsulfonyl fluoride to enable immunoblotting. The primary antibodies included rabbit anti-caspase-3 (1:1,000, 9662S, Cell Signaling Technology, MA, USA), rabbit anti-BOK (1:500) and mouse anti-β-actin (1:1,000, sc-47778, Santa Cruz Biotechnology, CA, USA). All antibodies were diluted with TBST buffer (0.1% Tween-20, 50 mM Tris-HCl, 150 mM NaCl, pH 7.4) and incubated at 4ºC overnight on PVDF membranes. Secondary antibodies conjugated with horseradish peroxidase (1:5,000, Abbkine Scientific Co. Ltd., US) were incubated for 90 min at ambient room temperature with the PVDF membranes. Signals were detected using an enhanced chemiluminescence reagent coupled to a Bio-Rad ChemiDoc MP instrument.

RT-PCR

Expression levels of mRNA of mouse DLX6-AS1, human DLX6-AS1, mouse β-actin and human β-actin (primer sequences are shown in Table 2). miR-149-3p and U6 levels in brain sections and cells were analyzed. The methodology is documented in the supplementary materials section.

Dual luciferase reporter gene assay

Assays were carried out as previously described [25]. Three binding sites on the DLX6-AS1 3′UTR were predicted to be regulated by miR-149a-3p (position1:98-106, position2:303-309, position3:490-498) and these sites were mutated via site-directed mutagenesis. The mutated (Mt) and wild-type (Wt) sequences of the 3'-UTR segments of DLX6-AS1 were inserted in to GV272 vectors (Genechem, Shanghai, China) to conduct luciferase reporter vectors. The sequence of miRNA-149-3p was inserted into the GV251 vectors (Genechem, Shanghai, China) to conduct miRNA-149-3p expression plasmid. For the reporter assays, the 293T cells were then placed in 24-well plates and 0.6 μg of GV251-mir-149-3p plasmid or the negative control was co-transfected with the 0.2 μg constructed DLX6-AS1 WT or MUT luciferase reporter vectors. The luciferase activity was measured using a luciferase assay kit (Promega, Madison, US) after transfection for 48 h according to the manufacturer's instructions. Experiments were repeated ≥ 3 times using different primary cells and plasmids.

Statistical analysis

Data are reported as the mean ± SEM after ≥ 3 replicate measurements and analyzed using a t-test, one-way ANOVA and Tukey’s test. Statistical analyses were performed using Graph Pad Prism (ver. 5.0) and a P-value < 0.05 was considered to be a significant finding.

Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. This article does not contain any studies with human participants performed by any of the authors.