Cell Culture and Transfection

Immortal cell lines were acquired from the American Type Cell Culture Collection (ATCC) and cultured at 37 °C in a humidifier containing 5% CO2. Both BE(2)-M17 (CRL-2267) being simply referred to as M17 cells and SH-SY5Y (CRL-2266) cell lines were maintained in Opti-MEM (31,985,062; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) containing 5% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA). PC12 cells were maintained in low-glucose Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% heat-inactivated horse serum (Invitrogen) and 5% FBS (Invitrogen); 293T cells were maintained in DMEM (10,569,044; Gibco). Lipofectamine™ 3000 reagent (L3000015; Thermo Fisher Scientific) was used for the transfection of DNA constructs into cells according to the manufacturer’s instructions.

To obtain primary mouse brain fibroblasts (MBFs), the cerebral meninges samples of fetal mice were peeled under a microscope (Leica S8 APO; Wetzlar, Germany). These were then placed in a Petri dish with precooled phosphate-buffered saline (PBS) and digested with 0.25% trypsin at 37 °C for 20 min; the cell suspension was obtained by agitation. The cells were collected via centrifugation at 200–400 × g, and the pellet was resuspended in DMEM containing 10% FBS, 100 units/mL penicillin, and 100 µg/mL streptomycin. Thereafter, approximately 8 × 104/mL cells were seeded in a 3.5-cm Petri dish at 37 °C with 5% CO2. The medium was replaced every other day.

DNA Construct

The full-length DNA (3414 bp) coding LINC00894 (ENST00000449111.5) was amplified using PCR with the following primer pairs: LINC00894-F: 5′-GCGGCTAGCACTTGCCACAAGGAGACGCTG-3′ (Nhe1) and LINC00894-R: 5′-GCGGCGGCCGCTCCAAATAGGCACTAAATCCA-3′ (Not1); the fragment containing the Nhe1 and Not1 cloning sites was cloned into pcDNA3.1. EIF5 (NM_001969.5), ATF4 (NM_001675.4), FGF21 (NM_019113.4), and ACOD1 (NM_001258406.2) overexpression vectors were constructed by cloning the three open reading frames into pcDNA 3.1 vectors. CRISPR reagents were generated to target LINC00894 (designed as LINC00894 gRNA) using the following oligo pairs: LINC00894-gRNA4-F: 5′-CACCGAGGCAGGGTGTGCTGGGTCT-3′ and LINC00894-gRNA4-R: 5′-AAACAGACCCAGCACACCCTGCCTC-3′ (bases in bold typeface are gRNA sequences) and cloned into a lentiviral vector (lentiCRISPR; Addgene plasmid # 52,961). qRT-PCR was used to confirm that LINC00894 expression was silenced by LINC00894 gRNA. The FGF21 promoter DNA was amplified using PCR with the following primers: 5′-GACAAGGAGCGTGACCATTGAAGC-3′ and 5′-ATGGCTCGGGTCCTCAGGTGATCT-3′; ACOD1 promoter DNA was amplified using PCR with the following primers: 5′-CATAAGATGCCACAATTTGGTG-3′ and 5′-CGTTGTAAAGAAGAGGTTCAG-3′; the two promoter DNA fragments were cloned into the pGL4.0 luciferase report vector (Promega, WI, USA) using Gibson assembly cloning kits (E5510S, NEB, MA, USA).

Animals and MCAO/R Model

Healthy adult male Sprague–Dawley rats or male C57BL/6 mice aged 8 weeks were purchased from the Comparative Medicine Center of Yangzhou University, China. All experiments were conducted by following the “Guiding opinions on treating experimental animals” issued by the Ministry of Science and Technology and approved by the Animal Ethics Committee of Yangzhou University (Yangzhou, China; Approval No. 202,311,004 and No. 202,304,026).

Control recombinant adeno-associated virus (AAV) and LINC00894 RNA carrying AAV (pAAV-CMV-PGI-19,090,012-tWPA) were obtained from Heyuan Biotechnology Shanghai Company, China (www.oobio.com.cn). The animals were randomly divided into control and treatment groups, which were administered AAV (AAV-con) and AAV carrying LINC00894 (AAV-LINC00894), respectively. The animal was anesthetized by injecting Zoletil®50 into the abdominal cavity before being fastened to a brain stereotaxic apparatus (RWD Life Science, Shenzhen, China). Then the virus were intracerebroventricularly (icv.) injected into the left lateral ventricle of the animal (rats X: +3.0 mm, Y: +1.0 mm, Z: −3.0 mm; mice X: +1.0 mm, Y: +1.0 mm, Z: −2.0 mm); a dose of 2 µl of LINC00894 virus or control AAV virus (titer 7.72 × 1012) was injected into rats, while 0.8 µl of these virus (titer 7.72 × 1012) was administered to mice. The MCAO/R model was established after a month of virus administration, as previously described [37]. The animal was anesthetized by injecting Zoletil®50 (50 mg/kg) into the abdominal cavity, and the anterior midline skin was incised. Thereafter, the internal carotid artery (ICA), external carotid artery (ECA), and left common carotid artery (CCA) were separated. The distal end of the ECA was ligated, and the ECA and its branches were coagulated near the ligation point. A suture was inserted from the ECA through the CCA bifurcation into the ICA, with the arterial clamp on the ICA loosened and the suture inserted into the intracranial ICA segment. The blood flow was blocked for 1 h and the suture was retrieved for reperfusion for 24 h. Neurological behavioral tests were performed 24 h post-reperfusion. Longa neurological examination scores were used to assess neurological deficit, which was divided into six grades: 0 points, no neurological deficit; 1 point, failure to fully extend left forelimb, mild focal neurological deficit; 2 points, circling to the left, moderate neurological deficit; 3 points, falling to the left, severe focal deficit; 4 points, no spontaneous walking and depressed level of consciousness; 5 points, death. The average score was used to compare the behavior difference between LINC00894 virus or control AAV virus infected animal in MCAO model. At last, the infarct size was determined by staining with 2,3,5-triphenyltetrazolium chloride (TTC, Amresco LLC., 298-96-4) and was analyzed with Image J software (v1.50i).

Oxygen–Glucose Deprivation (OGD/R)

Cells were seeded into a six-well plate at 50% confluence and maintained in culture medium for 24 h before being washed thrice using PBS; they were then maintained in DMEM without glucose under 95% N2 and 5% CO2 for 4–16 h. Following oxygen deprivation treatment, the medium was replaced with a normal expansion medium, and the cells were maintained for another 1 h before the cells were used for further analysis. The DNA transfected cells were used 24 h after transfection for OGD challenge or other experiment.

Apoptosis Detection

Cellular apoptosis was evaluated using fluorescence-activated cell sorting with a PE–Annexin V apoptosis detection kit (cat:559,763, BD Biosciences, San Jose, CA, USA) according to the manufacturer’s instructions. Briefly, cell pellets in each treatment were obtained by centrifugation of the samples at 400 × g before being resuspended in binding buffer at 25 °C at a density of 1 × 106 cell/mL; the cell suspension was incubated with annexin-V–FITC and 7-AAD at 25 °C in the dark for 15 min. The cells were then detected using a flow cytometer (CytoFLEX, Beckman, San Jose, CA, USA), and the percentages of apoptotic cells in each group were analyzed using FlowJo (vX 10.0.7r2).

RNA–Protein Pull-Down and Electrophoretic Mobility Shift Assay (EMSA)

The DNA construct encoding LINC00894 was synthesized and cloned into pcDNA3.1. Sense and antisense LINC00894 were obtained using PCR before being synthesized using the MEGAscript™ T7 Transcription Kit (AM1333; Thermo Fisher Scientific). The total protein extract from SH-SY5Y was incubated with sense and antisense LINC00894 labeled with biotin using the Pierce™ Magnetic RNA-Protein Pull-Down Kit (#20,164; Thermo Fisher Scientific) according to the manufacturer’s instruction. The sense and antisense LINC00894 bound components were analyzed using mass spectrometry, and the unique peptide sequences enriched in the sense LINC00894 group were analyzed using immunoblotting.

To confirm if sense LINC00894 interacted with EIF5, we synthesized LINC00894 using the MEGAscript™ T7 Transcription Kit (AM1333; Thermo Fisher Scientific); approximately 5 pmol/L LINC00894 was labeled using T4 polynucleotide kinase and [γ32-P] ATP. A 30-µL EMSA reaction mixture containing recombinant EIF5, 100 mM KCl, 1 µg poly (dI–dC), 0.033 mm ZnCl2, and ∼40 fmol labeled LINC00894 was incubated on ice for 20 min. Protein–RNA complexes were resolved using 5% polyacrylamide gel electrophoresis (PAGE) without sodium dodecyl sulfate at ∼130 V for 2 h at 4 °C; the gels were then dried, and the protein–RNA complexes were visualized using autoradiography.

RNA Immunoprecipitation Assay

For RNA immunoprecipitation, 1.5 × 107 SH-SY5Y cells were washed thrice with cold PBS and scraped into 1 mL of PBS. The cells were then centrifuged and lysed using RIPA buffer (Merck Millipore, MA, USA). The protein A/G magnetic beads were pre-bound with 6 µg EIF5 antibodies or IgG in immunoprecipitation buffer (140 mM NaCl, 20 mM Tris-HCl pH 7.5, 0.05% TritonX-100) for 2 h before being incubated with 100 µL of cell lysates overnight at 4 °C with agitation. The magnetic beads were washed, and the bound RNA was eluted with 400 µL of elution buffer for 2 h. The eluted RNA was precipitated with ethanol and dissolved in RNase-free water. Enrichment of certain fragments from the IgG control or EIF5 groups was determined using real-time PCR with the following primer pair: sense: 5′-AGCAGACCATGAGAGGGAGT-3′, antisense: 5′-CCTCTAGTGGGCAACCCTTG-3′. The antibodies used in this experiment were as follows: EIF5 (Thermo Fisher Scientific; A301-771 A) and Anti-IgG (Cell Signaling Technology, MA, USA; #2729).

RNA Isolation and qRT-PCR

Cellular total RNA was obtained from cells using the TRIzol reagent (Invitrogen). RNA quality was evaluated using electrophoresis and the ratio of OD 260/OD 280. For cDNA synthesis using reverse transcription with the PrimeScript RT Reagent Kits (Takara Bio, Kusatsu, Japan), 1000 ng of RNA was used. cDNA was used as the template and was amplified in triplicate using qRT-PCR with the CFX Connect Real-Time PCR Detection System (Bio-Rad, CA, USA) and SYBR Premix Ex Taq (Takara Bio) according to the manufacturer’s instructions. All primers used are shown in Table 1. The qRT-PCR cycling conditions were as follows: initial denaturation at 95 °C for 45 s, 95 °C for 35 s, and annealing at 60 °C for 35 s for 40 cycles. The 2−ΔΔCT method, with β-actin as the internal control, was used to determine the relative expression. Fluorescent signals were measured after each primer-annealing step at 60 °C.

Table 1 Primers used in the qRT-PCRRNA-Seq

The bulk RNA-seq analysis (contract ID: 80-1220563257) was supported by AZENTA Company, Suzhou, China. The libraries for sequencing were generated from the total RNA of rat brains infected with either AAV-con or AAV-LINC00894. Approximately, 1 µg total RNA was used for library preparation.

The oligo(dT) beads were used to isolate poly(A) mRNA. First-strand cDNA and second-strand cDNA were synthesized using random primers. The purified double-stranded cDNA was then treated to repair both ends, and a dA-tail was added in one reaction, followed by T-A ligation to add adaptors at both ends. Size selection of adaptor-ligated DNA was then performed using DNA clean beads. Each sample was then amplified using PCR with P5 and P7 primers, and the PCR products were validated. Libraries with different indices were multiplexed and loaded on an Illumina HiSeq instrument (Illumina, CA, USA) and sequenced using a 2 × 150 paired-end (PE) configuration according to the manufacturer’s instructions.

Western Blot Analysis

Cells attached in the culture wells were lysed for 30 min at 4 °C with RIPA buffer (Merck Millipore) containing 1 mM sodium orthovanadate, 100 mM NaCl, 2.5 mM Tris-HCl (pH 7.5), 10 µg/mL leupeptin, and 10 µg/mL aprotinin. The supernatant was collected from the cell lysis solution by centrifugation at 13,500 × g for 15 min at 4 °C and the protein concentration was measured using the Lowry protein assay. Thereafter, 15–80 µg protein was separated using PAGE and electro-transferred to a polyvinylidene fluoride membrane. The membrane was blocked by incubation with 5% nonfat milk powder in Tris-buffered saline containing tween 20 (TBST) for 2 h at 25 °C before incubation with primary antibodies at 4 °C overnight. Horseradish peroxidase-conjugated anti-mouse (1:5000; ORIGENE, China) or anti-rabbit immunoglobulin IgG (1:5000; ORIGENE) was used as the secondary antibody, and the membrane was incubated for 2 h at 25 °C. Immunoreactive bands were visualized via enhanced chemiluminescence (ECL; Bio-Rad). For quantification, the ECL signals were digitized using the Image J software (v1.50i). The antibody used were anti-EIF5 antibody (#ab170915, Abcam), anti-β-Actin antibody (#A5441, Sigma-Aldrich), anti-GAPDH antibody (#ab181602, Abcam), monoclonal antibody recognizing Ubiquitin (P4D1) (#3936, CST), Anti-ATF4 (#11,815, CST), Cleaved Caspase-3 Antibody (#9661,CST), Caspase-3 Antibody (#9662,CST), Anti-GCLC antibody (#ab207777, Abcam), Anti-IRG1 antibody (#ab222411, Abcam), Anti-FGF21 antibody (#ab171941, Abcam). All original gel/blot images are provided in Additional File 1.

Luciferase Reporter Assay

Cells were plated in 24-well plates for 24 h and then transfected with luciferase vectors (200 ng) with or without 50 ng of ACOD1 or FGF21 expressing vector (when necessary) using Lipofectamine 3000 Reagent or jetPRIME® (Polyplus Transfection, Strasbourg, France). The phRLMLP Renilla luciferase expression vector was co-transfected at 40 ng for each well to evaluate transfection efficiency. The cells were not lysed until 24 h post-transfection, and luciferase activity was determined using the dual luciferase reporter assay system (Promega, WI, USA). The relative promoter activity was calculated as a normalized firefly/Renilla ratio.

ChIP Assay

BE(2)-M17 neuroblast cell line (M17) cells fixed in formaldehyde were added to the culture medium at a final concentration of 1% and maintained in 10-cm culture plates at 25 °C. The cells were shaken for 10 min before adding glycine (0.125 M). After 10 min, the cells were washed twice with cold PBS, centrifuged at 500 × g, and lysed in SDS lysis buffer containing 1 mM phenylmethylsulfonyl fluoride, 2 mg/mL pepstatin A, and 2 mg/mL aprotinin. Sonication was used to break the DNA into 500–1000 bp fragments. The chromatin was incubated with agarose beads containing control anti-serum or anti-ATF4 antibody (rabbit mAb, CST11815) overnight at 4 °C. The agarose beads were subsequently pelleted and washed once with a low-salt wash buffer, high-salt wash buffer, and LiCl wash buffer, and twice with TE buffer. Thereafter, the DNA bound with agarose beads and antibodies was recovered using phenol/chloroform extraction and ethanol precipitation. The eluted DNA was analyzed using PCR with the PCR products purified and sequenced.

Statistical Analysis

Data are presented as mean ± standard error of the mean. All statistical analyses were conducted using Prism 8 (Version 8.0.1; GraphPad Software, San Diego, CA, USA). Unless otherwise mentioned, Student’s t-tests were used for comparisons, and results with P < 0.05 were considered significant.