Between January 2019 and October 2019, blood samples were collected from 30 patients who had suffered a cerebral ischemic stroke (female to male patient ratio, 17:13; aged 25–65 years) and 30 healthy individuals who were recruited to The Affiliated Hospital of Yangzhou University (Yangzhou, China). The study was approved by the Ethics Committee of The Affiliated Hospital of Yangzhou University (approval no. 2018-004-01) and all patients provided written informed consent prior to participation in the study. The inclusion criteria were as follows: Patients were admitted within 48 h from the onset of stroke, and diagnosed by cerebral imaging and a neurologist. The exclusion criteria were as follows: Intracranial hemorrhage, hematological diseases, pregnancy, cancer, severe renal failure, severe liver failure, recent myocardial infarction and ongoing treatment with anti-inflammatory drugs (20). To obtain the plasma samples, 2 ml of blood was collected in S-Monovette^® EDTA-KE tubes (Sarstedt, Inc.), which was subsequently centrifuged at 2,000 × g for 1 min at 4°C. The supernatant was transferred into a new tube and further centrifuged at 4,000 × g for 5 min at 4°C. The plasma was then acquired and stored at −80°C until required for further analysis.

PC12 cells cultured under OGD/R conditions have been extensively studied as an in vitro model system for the identification of mechanisms of neuronal death following ischemic insult and for potential neuroprotective targets (21–23). In the present study, PC12 cells were purchased from The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences and cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Sigma-Aldrich; Merck KGaA), 100 µg/ml streptomycin and 100 U/ml penicillin. Cells were maintained under humidified conditions with 5% CO₂ and 95% air at 37°C.

To establish the OGD/R model, PC12 cells were maintained in glucose-free DMEM supplemented with 10% FBS and streptomycin/penicillin mixture in an oxygen-free incubator with 5% CO₂ and 95% N₂ at 37°C for 2 h. Following hypoxic exposure, the cells were incubated in normal medium containing FBS and streptomycin/penicillin in an atmosphere containing 95% air and 5% CO₂ at 37°C for 12 h. PC12 cells in the control group were incubated under normoxic conditions containing 95% air and 5% CO₂ at 37°C for 24 h.

Knockdown of AK139328 expression was performed using short hairpin RNA (shRNA) targeting AK139328 (shRNA-AK139328-1; 5′-CACCGGAAACTCAGCTATCACATGCCGAAGCATGTGATAGCTGAGTTTCC-3′), shRNA-AK139328-2 (5′-CACCGCAGCAGAAAGACATGTTTGGCGAACCAAACATGTCTTTCTGCTGC-3′) and corresponding scrambled negative control (shRNA; 5′-CACCTTCTCCGAACGTGTCACGTTTCAAGAGAACGTGACACGTTCGGAGAATTTTTTG-3′), which were both synthesized by Shanghai GenePharma Co., Ltd. PC12 cells were transfected with 500 ng/µl shRNA-AK139328-1/2 or shRNA using Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The cells were harvested for use in subsequent experiments at 48 h post-transfection.

MTT assay was performed to analyze cell viability. PC12 cells were plated into 96-well plates (5×10³ cells/well). Following 24 h of incubation at 37°C, cells received OGD/R treatment as described above. Then, 20 µl MTT solution (5 mg/ml) was added into each well and incubated with the cells for 4 h at 37°C. MTT solution was subsequently discarded and 150 µl DMSO was added to each well for 10 min at room temperature to dissolve the purple formazan crystals. The optical density of each well was measured using a microplate reader at a wavelength of 570 nm.

Total RNA was extracted from PC12 cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The concentration and quality of RNA were assessed using a NanoDrop™ 3000 spectrophotometer (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Total RNA was reverse transcribed into cDNA using a PrimeScript RT Master mix (Takara Bio, Inc.) according to the manufacturer's protocol. qPCR was subsequently performed using a SYBR Premix ExTaq kit (Takara Bio, Inc.) with the following reaction conditions: 95°C for 2 min, followed by 40 cycles of 95°C for 20 sec and 65°C for 40 sec. The primer sequences were as follows: lncRNA-AK139328 forward, 5′-CCAGTTCTTGGTCCTGGTGT-3′ and reverse, 5′-GTGTCTGCAACCCGATAGGT-3′; GAPDH forward, 5′-AGCCACATCGCTCAGACAC-3′ and reverse, 5′-GCCCAATACGACCAAATCC-3′. The relative expression levels of the target genes were calculated using the 2^−∆∆Cq method (24). GAPDH was used as the internal reference gene, and the relative expression level was normalized to GAPDH.

PC12 cells were plated in the 96-well plates (5×10³ cells/well) for incubation for 24 h, followed by exposure to OGD/R. Then, the culture medium was collected and centrifugated for 5 min at 12,000 × g and 4°C to obtain the supernatant of PC12 cells. The levels of TNF-α, IL-1β and IL-6 in the supernatant of PC12 cells were measured using the corresponding ELISA kits (cat. no. 210-TA-005 for TNF-α; cat. no. 201-LB-005 for IL-1β; cat. no. S6050 for IL-6; R&D Systems, Inc.) according to the manufacturers' protocols. A ROS assay kit (cat. no. JL13783; Shanghai Jianglai Biological Technology Co., Ltd.) was used to evaluate the intracellular ROS levels in OGD/R-treated PC12 cells. Each experimental condition was plated five times and all assays were independently repeated three times.

Flow cytometry was performed to determine the induction of cell apoptosis using an Annexin V-FITC/PI Apoptosis Detection kit (Sigma-Aldrich; Merck KGaA). After the transfected PC12 cells were treated with OGD/R as described above, the cells were harvested, resuspended in 0.5 ml PBS and incubated with 5 µl Annexin V-FITC for 15 min and 10 µl PI (10 mg/ml) in the dark for 5 min. Apoptotic cells were analyzed using a FACScan flow cytometer (BD Biosciences). The data were analyzed using flow cytometry software (iSort™ Automated Cell Sorter; version A.0; Thermo Fisher Scientific, Inc.). The cell apoptosis rate calculated as the percentage of early and late apoptotic cells. The experiments were performed in triplicate.

Transfected PC12 cell slides were fixed with 4% paraformaldehyde at room temperature for 20 min and blocked with 5% skimmed milk at room temperature for 1 h. Subsequently, the cells were incubated with an anti-microtubule associated protein-2 (MAP-2; 1:1,000; cat. no. ab75713; Abcam) or anti-growth associated protein (GAP)-43 primary antibody (1:500; cat. no. ab75810; Abcam) overnight at 4°C. Following the primary antibody incubation, slides were incubated with Alexa Fluor^® 594-conjugated secondary antibody (1:1,000; cat. no. A-11012; Invitrogen; Thermo Fisher Scientific, Inc.) for 1 h at 37°C. Cell nuclei were counterstained with DAPI for 5 min at room temperature. Stained cells were observed under a laser confocal microscope (Olympus Corporation; magnification, ×200).

Total protein was extracted from transfected OGD/R-treated cells using RIPA lysis buffer (Beyotime Institute of Biotechnology). Total protein was quantified using a BCA assay kit (Beyotime Institute of Biotechnology) and protein samples (20 µg/lane) were separated via 10% SDS-PAGE. The separated proteins were subsequently transferred onto PVDF membranes and blocked with 5% non-fat milk at room temperature for 1 h. The membranes were then incubated with the following primary antibodies overnight at 4°C: Anti-endothelial nitric oxide synthase (eNOS; 1:1,000; cat. no. ab5589; Abcam), anti-MAP-2 (1:1,000; cat. no. ab32454; Abcam), anti-GAP-43 (1:1,000; cat. no. ab75810; Abcam), anti-Bcl-2 (1:1,000; cat. no. ab196495; Abcam), anti-Bax (1:1,000; cat. no. ab182733; Abcam), anti-cleaved caspase-3 (1:5,000; cat. no. ab214430; Abcam), anti-caspase-3 (1:2,000; cat. no. ab184787; Abcam) and anti-GAPDH (1:2,500; cat. no. ab9485; Abcam). Following the primary antibody incubation, the membranes were washed twice with TBS with 0.05% Tween-20 (TBST) and incubated with goat anti-rabbit IgG H&L secondary antibody (1:2,000; cat. no. ab6721; Abcam) for 1 h at room temperature. GAPDH was used as the internal loading control. Protein bands were visualized using ECL substrate (Pierce; Thermo Fisher Scientific, Inc.) and analyzed with ImageJ software (version 3.0; National Institutes of Health).

Statistical analysis was performed using SPSS 21.0 software (IBM Corp.) and GraphPad Prism 6.0 software (GraphPad Software, Inc.). Statistical differences between two groups were determined using an unpaired Student's t-test, while a one-way ANOVA followed by a Tukey's post hoc test was used to determine statistical differences among multiple groups. Data are presented as the mean ± SD. All experiments were repeated at least three times. P<0.05 was considered to indicate a statistically significant difference.

To investigate the role of lncRNA AK139328 in cerebral ischemic stroke, the expression levels of AK139328 were first analyzed in clinical specimens. AK139328 expression levels were significantly upregulated in the plasma of patients who had experienced a cerebral ischemic stroke compared with those noted in healthy individuals (Fig. 1A). In addition, AK139328 expression levels were determined in an in vitro OGD/R model. The RT-qPCR results revealed a significant upregulation in AK139328 expression levels in OGD/R-induced PC12 cells compared with cells cultured under normoxic conditions (Fig. 1B). These data indicated that AK139328 may participate in the pathophysiology of cerebral IRI.

To determine the effects of AK139328 on PC12 cells induced with OGD/R, AKI39328 expression was knocked down in PC12 cells and the transfection efficiency was evaluated using RT-qPCR (Fig. 2A). The results indicated that AK139328 expression levels were significantly downregulated in cells transfected with shRNA-AK139328-1 or shRNA-AK139328-2 compared with the control and shRNA-transfected cells. Moreover, the expression levels of AK139328 were lowest in shRNA-AK139328-1-transfected cells; therefore, shRNA-AK139328-1 was selected for use in subsequent experiments. The results of the MTT assay revealed that, after OGD/R induction, cell viability was significantly decreased compared with the control group, while silencing of AK139328 partially alleviated the OGD/R-induced loss in cell viability (Fig. 2B). In addition, the expression levels of Netrin-1 were significantly downregulated by OGD/R treatment compared with the control group; however, this downregulation was partially reversed following the knockdown of AK139328 (Fig. 2C).

The effects of AK13928 on inflammatory injury were subsequently determined. The results obtained from the ELISAs revealed that the protein levels of TNF-α, IL-1β and IL-6 in PC12 cells following exposure to OGD/R were significantly elevated compared with the control cells, while the production of these inflammatory factors was inhibited following transfection of the cells with shRNA-AK139328-1 in the presence of OGD/R (Fig. 2D). These results suggested that the knockdown of AK139328 may play an inhibitory role over the inflammatory responses induced by OGD/R.

Subsequently, the effects of AK139328 silencing on the induction of oxidative stress were determined in OGD/R-treated PC12 cells. OGD/R treatment significantly increased the production of ROS compared with control cells, whereas the concurrent transfection with shRNA-AK139328-1 partially reduced the increased levels of ROS in PC12 cells (Fig. 3A). In addition, the protein expression levels of eNOS were analyzed by western blotting. OGD/R injury significantly downregulated the protein expression levels of eNOS compared with the control group, while AK139328 silencing upregulated eNOS protein expression levels in OGD/R-injured PC12 cells (Fig. 3B). These findings suggested that the knockdown of AK139328 expression may alleviate OGD/R-induced oxidative stress levels in PC12 cells.

The effects of AK139328 silencing on the induction of OGD/R-injured PC12 cell apoptosis were determined to investigate the role of AK139328 in cerebral IRI. Flow cytometry data demonstrated that OGD/R significantly increased PC12 cell apoptosis compared with control cells, while AK139328 silencing decreased the apoptotic rate in OGD/R-treated PC12 cells (Fig. 4A). Moreover, western blotting analysis revealed that Bcl-2 expression levels were significantly downregulated, while the expression levels of Bax and cleaved caspase-3/caspase-3 were significantly upregulated following the induction of OGD/R compared with control cells. However, transfection with shRNA-AK139328-1 exerted inhibitory effects on cell apoptosis by partially reversing the trends in the expression levels of Bcl-2, Bax and cleaved caspase-3 observed in OGD/R-treated PC12 cells (Fig. 4B). These data indicated that the knockdown of AK139328 may exert suppressive effects in OGD/R-mediated PC12 cell apoptosis.

The effects of AK139328 on the neurite outgrowth of PC12 cells injured by OGD/R were analyzed. The results from the immunofluorescence staining demonstrated that MAP-2 and GAP-43 expression levels were increased in cells cultured under normal conditions, while the expression levels were slightly decreased by OGD/R treatment (Fig. 5A and B). Notably, the transfection with shRNA-AK139328-1 rescued OGD/R-induced decreased expression levels of MAP-2 and GAP-43 in OGD/R-induced PC12 cells. In addition, the protein expression levels of MAP-2 and GAP-43 were significantly downregulated in OGD/R-treated PC12 cells, which were subsequently reversed by AK139328 silencing (Fig. 5C). These data suggested that AK139328 silencing may protect neurite outgrowth in PC12 cells following the induction of OGD/R.