Long non-coding RNA HOXA11-AS regulates ischemic neuronal death by targeting miR-337-3p/YBX1 signaling pathway: protective effect of dexmedetomidine

Cell I/R stress model establishment and drug treatment

Neuro-2a cells were obtained from Cell Bank Chinese Academy Science (CAS, Shanghai, China). Cells were cultured in Dulbecco’s medium (DMEM, Gibco, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS), L-glutamine, 100 IU/ml penicillin, and 100 μg/ml streptomycin in a 37°C incubator with a humidified atmosphere of 5% CO₂. Dex (TOYOBO, Shanghai, China), dissolved in DMEM, was added into cell cultures at the doses of 5, 10, and 20 μM for 24 hours, and the dose of 10 μM was also applied for 3, 6, 12, and 24 hours to test its toxicity. The cells received equal volume of DMEM only were considered as the control group. Cell I/R stress model was built by oxygen and glucose deprivation as previous literature [30]. Glucose-free DMEM and phosphate-buffered saline (PBS) were bubbled with 100 % N₂ for 30 min to exclude oxygen. After washing twice with buffer, Neuro-2a cells were seeded and cultured in glucose-free DMEM for 3 hours, and then treated with perfusion for 24 hours.

MTT assay was used to detect cell viability. Briefly, neuro-2a cells were seeded in 96-well tissue culture plates at the density of 1 × 10⁴ cells per well for 24 hours. Cells were treated with the drug at designed time and dose. Then, 0.5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was added into the culture medium for a further 3 hours’ incubation to detect cell viability. The blue formazan products were dissolved in dimethyl sulfoxide (DMSO) and the optical density values at 490 nm (OD₄₉₀) were measured with a microplate reader.

Quantification of DNA fragmentation

DNA fragmentation was quantified using a cellular DNA fragmentation enzyme-linked immunosorbent assay (ELISA) kit (Millipore-Sigma, Billerica, MA, USA) according to the manufacturer’s protocol. Briefly, Neuro-2a cells labeled with bromodeoxyuridine (5-bromo-2’-deoxyuridine) (BrdU) were subcultured in 24-well tissue culture plates at a density of 2 × 10⁵ cells per well overnight. After harvested and suspended in culture medium, one hundred microliters were added to each well of 96-well tissue culture plates. After drug treatment, cells were seeded at 37°C in a humidified atmosphere of 5% CO₂. Amounts of BrdU-labeled DNA in the cytoplasm were detected with anti-BrdU antibody conjugated with peroxidase. DNA fragmentation was quantified as optical density values at an optimal wavelength of 405 nm using a microplate photometer (GloMax^®-96 Microplate Luminometer, Promega).

Analysis of apoptotic cells

Cell apoptosis was quantified using propidium iodide (PI) and Annexin V staining. After Dex and I/R treatments, Neuro-2a cells were subjected to flow cytometric analysis to detect apoptosis using the Annexin V-fluorescein isothiocyanate (FITC)/PI Apoptosis Kit (BD Biosciences, San Jose, CA, USA) according to the manufacturer’s protocol. Briefly, after washing with PBS and the binding buffer for one time each, cells were stained with Annexin V-FITC/PI for 20 min at room temperature in dark. After washing with the binding buffer once, the labeled cells were detected immediately by a flow cytometer (Beckman Coulter, Franklin Lakes, NJ, USA). The percentage of apoptotic cells was defined as the % of Annexin V-positive cells (including both PI-negative and PI-positive populations) among the whole cells.

RNA extraction and qRT-PCR analysis

Total RNA and miRNAs were extracted from brain infarction tissues, or brain tissues after HOXA11-AS or Dex injection, or differently conditioned neuro-2a cells with or without transfection of si-HOXA11-AS, PcDNA3.1-HOXA11-AS (Gene-Pharma, Shanghai, China) using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and miRNeasy Mini Kit (Qiagen, Hilden, Germany), respectively. For miRNA analysis, cDNA was obtained using the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) and qRT-PCR was performed using TaqMan miRNA assay kit (Applied Biosystems). U6 small nuclear RNA was used as an endogenous control for normalization. For mRNA analysis, cDNA was synthesized by using M-MLV reverse transcriptase (Invitrogen) and reverse transcription primers Oligo (dT). qRT-PCR was then performed with SYBR Green Real-Time PCR Master Mixes (Thermo-Fisher, Waltham, MA, USA) on a PCR machine (Applied Biosystems).

Luciferase reporter assay

The targeting relationship between HOXA11-AS and miRNAs, miR-337-3p targeted candidate genes and the binding sites of miR-337-3p were predicted using the online tools miRDB (https://mirdb.org/), TargetScan (http://www.targetscan.org/), and microT (https://dianalab.e-ce.uth.gr/html/dianauniverse/index.php?r=microT_CDS). Before luciferase reporter assay, pmirGLO- HOXA11-AS-WT, and pmirGLO-HOXA11-AS-MUT were constructed by GenePharma (Gene-Pharma, Shanghai, China). miR-337-3p mimics, mimic control, miR-337-3p inhibitor and a negative control of scramble RNA were also from GenePharma (Gene-Pharma, Shanghai, China). PcDNA3.1-HOXA11-AS, Si-HOXA11-AS and si-s-HOXA11-AS plasmids were designed and cloned into a pcDNA3.0 vector. Luciferase activity was measured when cells were harvested 48 hours post-transfection with the Dual Luciferase Reporter Assay System (Promega, Madison, WI, USA) according to the manufacturer’s instructions.

Western blot

Cultures cells or brain samples were lysed and quantified. Protein samples (20 μg) were separated by 10% sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and then transferred to polyvinylidene difluoride (PVDF) membranes. After blocking for 2 hours with 5% bovine serum albumin (BSA) in Tris Buffered Saline with Tween 20 (TBST) and 0.1% Tween 20, primary antibodies against Ybx1 and β-actin (1:1000, Miliporesigma, Billerica, MA, USA) were subsequently added and incubated with the membrane for 16 hours at 4°C. After washing 3 times with TBST, the secondary antibody (1:5000 dilution; Miliporesigma, Billerica, MA, USA) was added to the membrane and the membrane was incubated for 1 hour at room temperature. Protein bands were detected by enhanced chemiluminescence and analyzed by Image-J software.

Animals and middle cerebral artery occlusion (MACO) model

C57BL/6 J male mice (3 months old, 25–28 g) were obtained from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) and kept in a humidity and temperature-regulated room with the cycle of 12-h light and 12-h dark. The anesthetized animal was placed under an operating microscope to make a midline incision. Right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were exposed. A 6/0 surgical nylon monofilament with a rounded tip was induced into the lumen of ICA gently from the right ECA until the rounded tip blocked the origin of the middle cerebral artery (MCA). After 60 min of MCAO, the surgical nylon monofilament was gently retracted to allow reperfusion for 24 hours. The sham-operated animals were treated with similar operations except without surgical nylon monofilament insertion. Mice were kept on a heating panel which maintained the temperature at 37°C during operation. TTC (2,3,5-triphenyltetrazolium chloride) staining was performed for visualization of the brain infarction, as previously reported [31]. The infarct volume of I/R injury and the volume of the whole brain were determined. The infarct area (%) was calculated according to the ratio of infarct volume to the whole volume of brain. The use and care of animals were performed in accordance with the guidelines for the Care and Use of Laboratory Animals of Xijing Hospital, Fourth Military Medical University and approved by the Institutional Animal Care and Use Committee.

Stereotaxic injection of HOXA11-AS mimics in mice and drug administration

Mice in the HOXA11-AS group were anesthetized and fixed to a stereotaxic apparatus. HOXA11-AS mimics was diluted with the same volume of in vivo transfection reagent (Entranster™-in vivo; Engreen, Beijing, China) and administered into the lateral ventricle of mice. The stereotaxic coordinates were as follows: 0.6 mm posterior to the bregma, 1.0 mm lateral to the midline, and 2.5 mm under the dura. After one day injection, MCAO operation was conducted. Mice in the Dex group were administrated with Dex diluted with physiological saline.

Modified neurological severity score test

Neurological function was assessed using the modified neurological severity score (mNSS) method [32]. The tests were conducted by observers who were blinded to the experiments. Neurological function including motor, sensory, balance and reflex tests was graded on a scale of 0 to 18 (normal score, 0; maximal deficit score, 18). 1 score point was awarded for the inability to perform the test or for the lack of a tested reflex; thus, the higher score, the more severe was the injury.

Corner test

The corner test was performed to detect unilateral abnormalities of sensory and motor functions in the MACO mouse model, largely following the previous report [33]. The mouse was places in two pieces of 30 cm × 20 cm × 1 cm wooden board. The two wooden boards were placed at 30° angle, and there was a small gap between the two boards. The mouse was encouraged to enter this corner and was placed facing the corner. The mouse moved forward or upward and eventually turned left or toward turning right back. Cerebral ischemia mice preferentially turned back to the non-injured side, ipsilateral (right) side each time. Numbers of corners visited were recorded during 300 seconds.

Statistical analysis

Data were expressed as mean ± standard deviation (SD). SPSS software (version 20; IBM, Armonk, NY, USA) and one-way Analysis of Variance (ANOVA) were used for statistical analysis. A P value smaller than 0.05 was considered statistically significant.

Availability of data and materials

The datasets used and/or analyzed during the present study are available from the corresponding author upon reasonable request.

Dex protected Neuro-2a cells from I/R-induced apoptotic death

Neuro-2a cells were treated with Dex at the doses of 5 μM, 10 μM, and 20 μM for 24 h, or at the dose of 10 μM for 3, 6, 12, 24 h to evaluate drug toxicity. The results showed that the cells viability had no much changes, indicating that Dex had no cytotoxicity in these doses (Figure 1A) and time range (Figure 1B). Neuro-2a cells were exposure to OGD for 3 h and then subjected to reperfusion for 24 h (OGD/R) significantly decreased cell viability by about 40% (Figure 1C). To determine the effects of Dex on I/R-induced insults to neuronal cells, Neuro-2a cells were treated with 10 μM Dex before ischemia for 3 h then received reperfusion for 24 h (I/R). Dex rescued cell viability, which increased from 60.5% of control in the OGD/R alone group to 81.8% of control in the OGD/R+Dex group (Figure 1C). In addition, Neuro-2a cells exposed to I/R led to a 2.6-fold stimulations of DNA fragmentation, and additional administration of Dex deceased I/R-induced DNA fragmentation by 44.5% (Figure 1D). Moreover, exposure to I/R caused cell apoptosis and additional Dex treatment lowed the percentages of apoptotic cells from 44.9% to 12.9% (Figure 1E). Thus, Dex could protect Neuro-2a cells from I/R-induced apoptotic cell death.

Figure 1. Protective effects of dexmedetomidine (Dex) on ischemia/reperfusion (I/R)-induced neuronal death. (A) Neuro-2a cells were exposed to 5, 10, 20 μM Dex for 24 h, and cell viability was measured. (B) Neuro-2a cells were treated with 10 μM Dex for 3, 6, 12, and 24 h, and cell viability was measured. (C–E) The effects of Dex on I/R-induced insults. Neuro-2a cells were treated with 10 μM Dex, received ischemia for 3 h then reperfusion for 24 h (I/R). Cell viability was analyzed (C), and DNA fragmentation was quantified with an ELISA method (D). The apoptotic cells were quantified by Annexin V/PI staining and statistically analyzed (E). n = 3 for each group; ^*P < 0.05, compared with the Control group; ^#P < 0.05, compared with the OGD/R group.

Dex treatment protected OGD/R exposed Neuro-2a cells through up-regulation of HOXA11-AS expression and increased cell survival

The expression of HOXA11-AS was determined by quantitative real-time PCR in the cells after OGD/R. Dex treatment alone significantly up-regulated HOXA11-AS expression and OGD/R decreased OXA11-AS expression in Neuro-2a cells. Administration of Dex also inhibited the decrease of OXA11-AS expression induced by OGD/R (Figure 2A). To investigate the functional mechanisms of HOXA11-AS in OGD/R, we used sh-HOXA11-AS and pcDNA3.1-HOXA11-AS to down-regulate its expression (Figure 2B) and over-express HOXA11-AS (Figure 2C), respectively. Transfection of pcDNA3.1-HOXA11-AS upregulated HOXA11-AS expression in cells without or with exposure to OGD/R (Figure 2D). MTT results revealed that HOXA11-AS overexpression rescued cell viability of OGD/R-exposed Neuro-2a cells, compared with the OGD/R alone groups (Figure 2E). Transfection of pcDNA3.1-HOXA11-AS also decreased the percentage of apoptotic cells induced by OGD/R (Figure 2F). To further explore the effects of Dex and HOXA11-AS knockdown on cells viability and apoptosis, we administrated Dex and performed sh-HOXA11-AS transfection to OGD/R treated cells. Compared with the OGD/R alone group and the OGD/R+Dex group, the groups with additional down-regulated HOXA11-AS expression (Figure 2G) showed decreased cell viability (Figure 2H) and increased cell apoptosis in OGD/R treated cells (Figure 2I), indicating that Dex exerted protective effects on OGD/R exposed Neuro-2a cells at least partially through up-regulation of HOXA11-AS expression.

Figure 2. Dex treatment protected OGD/R-exposed Neuro-2a cells through up-regulation of HOXA11-AS expression and increased cell survival. (A) Neuro-2a cells with or without OGD/R were pre-treated with 10 μM Dex. The expression of HOXA11-AS was determined by quantitative real-time PCR. (B, C) Knockdown (B) and over-expression (C) of HOXA11-AS by transfection of sh-HOXA11-AS and pcDNA3.1-HOXA11-AS, respectively, were verified by quantitative real-time PCR. (D–F) Neuro-2a cells were transfected with control empty vector or pcDNA3.1-HOXA11-AS vector, and were left untreated or received OGD/R. Relative expression of HOXA11-AS (D), cell proliferation (E) and apoptosis of cells (F) in the indicated groups were quantitated. (G–I) Neuro-2a cells transfected with control sh-RNA or sh-HOXA11-AS were left untreated or treated with 10 μM Dex. Cells were then subjected to OGD/R. Relative expression of HOXA11-AS (G), cell proliferation (H) and apoptosis of cells (I) in the indicated groups were quantitated. n = 3 for each group; ^*P < 0.05, compared between the indicated groups.

HOXA11-AS competed binding to miR-337-3p to silence miR-337-3p and inhibit apoptosis

Previous studies have demonstrated that lncRNAs could be involved in different physiology and pathology processes by functioning as competing endogenous RNAs (ceRNAs) of specific miRNAs [5, 6, 11]. Thus, we attempted to investigate the potential interaction between HOXA11-AS and miRNA in OGD/R. We used online tools to predict miRNAs targeted by HOXA11-AS. We screened 8 candidate miRNAs in the Neuro-2a cells transfected with pcDNA3.1-HOXA11-AS, and found that miR-337-3p expression was significantly inhibited by HOXA11-AS overexpression (Figure 3A). HOXA11-AS transcript binding sites on miR-337-3p were analyzed by bioinformatics database starBase v2.0 (Figure 3B), and a dual-luciferase reporter assay was performed to confirm the potential interaction between HOXA11-AS and miR-337-3p. The results showed that miR-337-3p mimics significantly decreased the luciferase activity of Neuro-2a cells transfected with the luciferase reporter vector HOXA11-AS-WT, while no obvious change was observed in luciferase activity of cells transfected with HOXA11-AS-MUT vector (Figure 3C). Furthermore, relative expression of miR-337-3p in Neuro-2a cells transfected with pcDNA3.1-HOXA11-AS or sh-HOXA11-AS was detected by qRT-PCR. HOXA11-AS overexpression markedly decreased the expression of miR-337-3p in cells with or without OGD/R (Figure 3D). HOXA11-AS knockdown increased miR-337-3p expression in OGD/R treated cells, irrespective of the existence of additional Dex treatment (Figure 3E). As shown in Figure 3F, 3G, additional miR-337-3p overexpression through miR-337-3p mimics transfection significantly decreased cell viability and increased cell apoptosis in OGD/R exposed cells, compared to the cells received OGD/R alone. Taken together, HOXA11-AS competed binding to miR-337-3p to silence miR-337-3p and inhibit apoptosis, which was one of the mechanisms underlying Dex’s neuroprotection effects.

Figure 3. HOXA11-AS competed binding to miR-337-3p to silence miR-337-3p and inhibit apoptotic cell. (A) Neuro-2a cells were transfected with control empty vector or pcDNA3.1-HOXA11-AS vector, and the expression of candidate miRNAs was quantitated by quantitative real-time PCR. (B) Schematic diagram shows the HOXA11-AS transcript binding sites on miR-337-3p. The wild type (WT) and mutated (MUT) binding sites of HOXA11-AS are shown. (C) The luciferase activity of the reporter vectors was detected in Neuro-2a cells at 48 hours after co-transfection of the plasmid expressing wild type or mutant HOXA11-AS together with the NC mimic or the miR-337-3p mimic. (D, E) Neuro-2a cells were transfected with control empty vector or pcDNA3.1-HOXA11-AS vector, and were left untreated or received OGD/R (D). Neuro-2a cells transfected with control sh-RNA or sh-HOXA11-AS were left untreated or treated with 10 μM Dex. Cells were then subjected to OGD/R (E). The relative expression of miR-337-3p was quantitated. (F, G) Neuro-2a cells transfected with control miRNA mimics or miR-337-3p mimics were left untreated or received OGD/R. Cell proliferation (F) and apoptosis (G) in the indicated groups were measured. ^*P < 0.05, ^**P < 0.01, compared with the Control group or between the indicated groups.

Dex protected neuronal cells from I/R-induced apoptosis through regulating the downstream effector YBX1 of the HOXA11-AS/miR-337-3p axis

As shown in Figure 4A, Venn diagram shows the numbers of overlapping genes among the predicted genes using the online algorithms including miRDB, TargetScan and microT. We found that miR-337-3p might bind to 3′-UTR of Ybx1 mRNA by bioinformatics analysis (Figure 4B). Luciferase reporter assay was conducted to confirm Ybx1 was a direct target of miR-337-3p. The plasmids of Ybx1-WT or Ybx1-MUT was co-transfected with miR-337-3p mimics into Neuro-2a cells. The results showed that miR-337-3p mimics significantly decreased the luciferase activity of cells transfected with the Ybx1-WT vector, but not that of Ybx1-MUT (Figure 4C). Compared to the negative control group, the group with knockdown of miR-337-3p by miR-337-3p inhibitor had significantly decreased miR-337-3p expression (Figure 4D) and significantly increased Ybx1 protein expression (Figure 4E). Overexpression of miR-337-3p by miR-337-3p mimics decreased Ybx1 protein expression in both the cells with or without OGD/R, and the different was that OGD/R made the decrease much severely (Figure 4F). Under OGD/R, Dex and miR-337-3p inhibitor, administrated separately or together, increased Ybx1 protein expression in cells; however, the treatment with Dex and miR-337-3p inhibitor together resulted in a much more increase of Ybx1 protein expression (Figure 4G). In addition, Ybx1 overexpression (Figure 4H) enhanced cell viability (Figure 4I) and reduced cell apoptosis (Figure 4J) induced by OGD/R. These results showed that HOXA11-AS competed with Ybx1 mRNA for binding to miR-337-3p, and Dex protected neuronal cells from ischemia/reperfusion-induced apoptosis through regulating the downstream effector Ybx1 protein.

Figure 4. Dex protected neuronal cells from apoptosis induced by ischemia/reperfusion through regulating YBX1 expression. (A) Venn diagram shows the numbers of overlapping genes among the predicted genes of miR-337-3p using the online algorithms including miRDB, TargetScan and microT. (B) Schematic diagram shows the matching base pairs between miR-337-3p and the wild type (WT) or mutated (MUT) 3′-UTR of Ybx1 mRNA. (C) The luciferase activity of the reporter vectors was detected in Neuro-2a cells at 48 hours after co-transfection of the plasmid expressing wild type or mutant 3′-UTR of Ybx1 mRNA together with the NC mimic or the miR-337-3p mimic. (D, E) Neuro-2a cells were transfected with control miRNA inhibitor or miR-337-3p inhibitor, and the expression of miR-337-3p (D) and protein level of Ybx1 (E) were quantitated at 48 hours after transfection. (F) Neuro-2a cells were transfected with control miRNA mimics or miR-337-3p mimics, and left untreated or received OGD/R. The protein level of Ybx1 was quantitated. (G) Effects of miR-337-3p inhibitor transfection and Dex treatment, alone or in combination, in OGD/R-treated Neuro-2a cells on the expression of Ybx1 protein. (H) Confirmation of Ybx1 overexpression in Neuro-2a cells at 48 hours after transfection of Ybx1-overexpression (OE) plasmid. (I, J) Control cells or Ybx1-OE cells were left untreated or received OGD/R. Cell proliferation (I) and apoptosis (J) in the indicated groups were measured. ^*P < 0.05, compared with the Control group or between the indicated groups.

Dex/HOXA11-AS protected against ischemic damage and improved neurological deficits in vivo

To verify the mechanisms underlying the neuroprotective effects of Dex, we used Dex and HOXA11-AS mimics to treat mice in the model of ischemia stroke. Dex was intraperitoneally injected to mice and HOXA11-AS mimics was injected with in vivo transfection reagent into the lateral ventricle of mice before MCAO operation. The expression of HOXA11-AS was determined by quantitative real-time PCR in mouse brain 24 h after MCAO. Dex and HOXA11-AS mimics treatment increased HOXA11-AS expression, which was down regulated in MCAO mice brain, in comparison to the Sham group (Figure 5A). The brain infarction was detected by TTC staining to visualize the damaged regions for determination of the infarct volume of I/R injury. It showed that Dex and HOXA11-AS mimics treatment significantly decreased the percentages of area with I/R-induced brain infract, compared with the MACO group (Figure 5B). A neurological scoring system and corner test were performed to evaluate neurological deficits after I/R and the beneficial effects of drugs. We found a less neurological score in Dex and HOXA11-AS mimics injected mice, compared with the MACO mice (Figure 5C). It was also noteworthy that Dex and HOXA11-AS mimics injection resulted in better recovery, as evidenced by less right turn in corner test (Figure 5D), which was used to detect unilateral abnormalities of sensory and motor functions in this animal model. Moreover, the increase of miR-337-3p expression induced by MCAO was attenuated by Dex and HOXA11-AS mimics treatments (Figure 5E). Consistently, the decrease of Ybx1 level induced by MCAO was also reversed by Dex and HOXA11-AS mimics treatments (Figure 5F). Collectively, these results indicated that Dex and HOXA11-AS overexpression protected against ischemic damage and improved neurological deficits in MCAO mice model through regulating Ybx1 protein expression.

Figure 5. Dex/HOXA11-AS protected against ischemic damage and improved neurological deficits in vivo. (A) The expression of HOXA11-AS was determined by quantitative real-time PCR in mouse brain after MCAO and Dex/HOXA11-AS mimics treatments. n = 5 mice for each group. (B) Representative illustrations of cerebral sections by TTC staining. Infracted region was stained white. The infarction volume was evaluated as the percentage of infarct area relative to the whole brain. (C, D) mNSS score (C) and numbers of right turns in the Corner test (D) in the indicated groups were summarized. n = 5 mice for each group. (E) The expression of miR-337-3p was determined by quantitative real-time PCR in mouse brain after MCAO and Dex/HOXA11-AS mimics treatments. n = 5 mice for each group. (F) The levels of Ybx1 in mouse brain after MCAO and Dex/HOXA11-AS mimics treatments were determined by western blot. n = 3 for each group. ^*P < 0.05, compared between the indicated groups.