Cell culture and treatment
PC-12 cells were obtained from the Shanghai Institutes for Biological Sciences (Shanghai, China). Cells were cultured in Dulbecco modified Eagle medium (DMEM; Gibco, Garlsbad, CA) containing 10% fetal bovine serum and 1% penicillin/streptomycin at 37 °C in a 5% CO2 atmosphere.
PC-12 cells were incubated with 200 μM H2O2 for 24 h to construct a cell injury model, and the cells cultured in the same medium without H2O2 were used as negative controls.
Small interfering RNAs (siRNAs) directed against MIAT and RBFOX2, and negative control (NC) siRNA were synthesized by GenePharma (Shanghai, China). Adenovirus-mediated MIAT, RBFOX2, MCL-1S, and MCL-1L overexpression vectors were constructed by RuiBiotech (Beijing, China). Transfection of cells was carried out with lipofectamine ® 3000 reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. Cells were infected with Adenovirus-mediated vectors with a MOI of 20.
According to a previous study , a pair of MCL-1 SBOs was designed to target the splicing sites at the 3' and 5' ends of exon 2 of MCL-1L pre-mRNA, so that exon 2 could be spliced off, thus changing the splicing mode of MCL-1L pre-mRNA from MCL-1L to MCL-1S mRNA. All steric-blocking oligonucleotides (SBOs) were synthesized, and the Endo-Porter delivery system was purchased from Gene Tools (Philomath, OR, USA). The sequences of the SBOs at the 3' acceptor and 5' donor splice sites of Mcl-1 pre-mRNA exon 2 were 5'-CGAAGCATGCCTGAGAAAGAAAAGC-3' and 5'-AAGGCAAACTTACCCAGCCTCTTTG-3', respectively. The non-targeting oligonucleotide sequence was 5'-CCTCTTACCTCAGTTACAATTTATA-3' (Con-SBO). The delivery efficiency of the Endo-Porter system was optimized by using a fluorescence microscope to observe the number of fluorescent positive cells in a limited field of view.
Animals and treatments
Adult male Sprague-Dawley (SD) rats (weighing 200–220g) were obtained from Center for Animal Experiment of Henan province (Zhengzhou, Henan, China). Rats were housed in standard conditions with controlled temperature (24 ± 2 °C) with 12:12 light/dark cycle and freely fed and watered. Animal experiments performed in our study were approved by the Animal Ethics Committee of Hong Hui Hospital. Animals were randomly divided into 5 groups: Sham group, SCI group, SCI plus adenovirus vector group, SCI plus Ad-MIAT group, SCI plus Ad-MIAT and Ad-RBFOX2 group. The rat SCI model was established by using the improved Allen method. In brief, rats were anesthetized by intraperitoneal injection of 10% chloral hydrate (3 mL/kg, Sino Chemical Reagent Company, Shanghai, China). Rats’ backs were shaved to expose the T9-11 spinous process and vertebral segments. Rats underwent laminectomy of vertebral T10, and then a 10 g rod (2.5 mm in diameter) was dropped from a height of 12.5 mm to the spinal cord to generate a moderate contusive injury in the SCI group. After injury, the spinal cord was washed with normal saline, the incision was sutured, and antibiotics were administered for three consecutive days. Rats in the sham group were only subjected to laminectomy. The empty vector, Ad-MIAT and Ad-RBFOX2 plasmids (200 µg of plasmids precipitated in 200 µL of PBS) were administered via tail vein injection immediately after SCI. At day 14 after SCI, a 10 mm long segment of the spinal cord centered at the injury epicenter was harvested for further examination.
Assessment of locomotor capacity
The assessment of athletic ability was based on the Basso-Beatie Bresnahan (BBB) exercise scale and the bevel test. All behavioral assessments were scored by three individuals who were blinded to grouping, and performed on days 1, 7, 14, 21 and 28 after SCI. The BBB test scores range from 0 to 21. The total score for severe neurological impairment was 0, while a total score of 21 indicated normal performance. The bevel test was performed on the test equipment. The maximum angle at which the rat remained in its position for more than 5 seconds without falling was recorded.
Preparation of spinal cord slices
Rats were deeply anesthetized with a lethal dose of sodium pentobarbital (80 mg/kg) and transcardially perfused with cold salt water, followed by 4% paraformaldehyde. Spinal tissues at the lesion site were dissected immediately after perfusion, immobilized overnight and cryopreserved in graded concentrations of sucrose (12%, 18%, and 24%). A 10 mm segment of the spinal cord including the injury epicenter was sectioned in transverse horizontal plane by using a cryostat (CM3050S, Leica, Wetzlar, Germany) at 10 or 20 μm thickness.
Hematoxylin-eosin (HE) staining and Nissl straining
For HE straining, sections were stained in hematoxylin solution for 5 min, washed in tap water, and soaked in 1% acid alcohol for 30 s. After washing again, the sections were stained in eosin for 30 s and dehydrated in graded concentrations of ethanol. For Nissl straining, sections were dewaxed with xylene and rehydrated in graded concentrations of ethanol. Next, sections were stained in Nissl staining solution (Solarbio, Beijing, China). Cells with typical neuronal morphology and nuclei were counted.
Fluorescent protein-LC3B dot assay
Cells were transfected with mRFP-eGFP-LC3B plasmid for 12 h. Then, Cells were treated in groups as shown in Figure 7. After 48 h, cells were fixed in 4% paraformaldehyde and washed in PBS. DAPI was used to stain nuclei. A confocal laser scanning microscope (Leica TCS SP8, Leica Biosystems, Wetzlar, Germany) was used to capture fluorescence images (400×), and the data were analyzed with Image J software.
Sections of the spinal cord were fixed, sealed, and incubated with the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) reaction mixture (Roche, Basel, Switzerland) at 37 °C for 1 h according to the manufacturer’s instructions. DAPI was used for counter staining. A fluorescence microscope was used to capture the images of the apoptotic cells. Total number of TUNEL-positive cells was counted from each slide and the averages were recorded to examine the numbers of apoptotic cells.
Cell apoptosis was measured with Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection Kit (BD Biosciences, San Diego, CA, USA). In brief, the treated cells were collected and resuspended in 100 μL of binding buffer. Next, the cells were cultured with 5 μL of Annexin V-FITC and 5 μL propidium iodide (PI) for 15 min in the dark. Fow cytometry was performed to measure the cell apoptosis, and the obtained data were analyzed using FlowJo software.
Cell viability assay
To evaluate the cell viability, the MTT assay was performed according to the manufacturer's instructions. Briefly, cells were seeded into 96-well plates with a density of 1 × 104 cells/well. After incubation for 48 h, 20 μL of MTT (Sigma-Aldrich, Shanghai, China) reagent was pipetted into each well, and cells were incubated for another 3 hours. Next, 200 μL of dimethyl sulfoxide solution (Sigma-Aldrich) was added, and the absorbance at 490 nm was measured with a microplate reader (Bio-Rad Model 550; Hercules, CA).
RNA pull‑down assay
RNA pull-down assay was performed by using a Pierce Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. The lysate of PC-12 cells were mixed with 50 pmol of biotinylated MIAT (MIAT probe) or NC probe, and incubated with 50 µL of streptavidin agarose magnetic beads (Life Technologies) at 4 °C for 1 h. The RNA-protein immunocomplex was eluted with Biotin Elution Buffer and then boiled in loading buffer. Western blotting was used to detect the RBFOX2 level.
RNA immunoprecipitation (RIP) assay
The Magna RNA immunoprecipitation kit (17-700, Millipore, Billerica, MA, USA) was used for RIP assay. The treated cells were collected and lysed in RIP lysis buffer, followed by incubation with magnetic beads which were pre-coated with anti-RBFOX2 or IgG (AP112, Sigma, St. Louis, MO, USA) at 4 °C for 6 h. qRT-PCR was performed to measure the MIAT level in the immunoprecipitation complex.
Total Protein was extracted from PC-12 cells and spinal cord tissues by using RIPA buffer with 1% PMSF (P0013B, Beyotime). BCA Protein Assay kit (P0012S, Beyotime) was used to measure protein concentrations. Next, equal amount of protein samples were subjected to SDS-PAGE and transferred to PVDF membranes. After blocking with 5% non-fat milk, the membranes were incubated with the following primary antibodies at 4 °C overnight: anti-Cleaved-caspase-3 (ab49822; 1:500; Abcam), anti-Cleaved-caspase-9 (ab2324; 1:500; Abcam), anti-RBFOX2 (ab264154; 1:3000; Abcam), anti-EXOC7 (ab118792; 1:3000; Abcam), anti-β-actin (ab8226; 1:1000; Abcam), and anti-MCL-1 (AHP998; 1:1000; Bio-Rad, USA), which can recognize both the MCL-1L (41 kDa) and MCL-1S (35 kDa) proteins. Then, the membranes were incubated with proper horseradish peroxidase (HRP)-conjugated IgG for 1 h at room temperature. The BeyoECL Plus (P0018S, Beyotime) was used to detect protein signals. The protein expression levels were determined according to the densitometry analysis using Image Lab software (Bio-Rad).
Total RNA was isolated from PC-12 cells and spinal cord tissues by using Trizol reagent following the manufacturer’s protocols. The samples were reversely transcribed by using the Bestar qPCR RT kit (DBI Bioscience, Ludwigshafen, Germany). qPCR amplification was carried out by using DBI Bestar ® SybrGreen qPCR MasterMix (DBI Bioscience) under the following conditions: 94 ℃ for 2 min, followed by 40 cycles of 94 ℃ for 20 s, 58 ℃ for 20 s, and 72 ℃ for 20 s. GAPDH was used as a endogenous control, and the relative transcript levels were calculated using the 2-ΔΔCT method.
Results in this study were presented as mean ± standard deviation. The comparison of more than two groups was conducted by one-way ANOVA test. The differences between two groups were investigated by Student’s t-test. P < 0.05 was considered to be a significant difference.