Experiment animals and grouping
Healthy adult SD rats weighed 250–280 g were purchased from the Shanghai Laboratory Animal Center, Chinese Academy of Sciences. For each animal experiment, 20 SD rats were used, which were divided into 4 groups (n = 5). All rats were housed in a terrarium with 22–26ºC, a 12 h light/dark cycle and ad libitum feeding and water. Rats were randomly divided into a model group and a sham operation group. The model group was further divided into MCAO/IR-12 h group, MCAO/IR-24 h group, and MCAO/IR-48 h (5 rats in each group). All experiments in this study were approved by All animal experiments were conducted according to the NIH Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of Fujian Medical University.
Construction of middle cerebral artery occlusion (MCAO)
The establish methods was referred to previous reports 13. All rats were anesthetized by 3% isoflurane (Shanghai Xinfan Biological Technology Co., ltd. Shanghai, China) in 70/30% medical air/oxygen at room temperature (25 ° C, 360 rag/ks). The median of neck was incised, the left external carotid artery (ECA) was ligated, the common carotid artery (CCA) and the internal carotid artery (ICA) were placed in the aneurysm clip. After breaking the ECA, a 0.25 mm (4 to 0) diameter nylon thread coated with 0.1% of L-polylysine at the end was inserted at a depth of 18–20 mm from the common carotid bifurcation. The animals were anesthetized again 2 hours later and the nylon thread was carefully pulled out. The rats of sham group were processed with a nylon thread insertion by depth of 15 mm and the remaining steps are the same. The anus temperature of the rats was maintained at 36.5 to 37.5 °C during the operation.
Construction of OGD/R model
In this experiment, the cell model of cerebral ischemia-reperfusion injury was simulated by means of glucose oxygen deprivation and re-glycemia and reoxygenation (OGD/R). PC12 cells were purchased from Shanghai Institute of Biosciences Cell Resource Center, Chinese Academy of Sciences. The specific manipulation steps are as follows. When the density reaches 80 to 90%, cells was transferred to a cell culture dish and cultured overnight until the cells were attached to the wall. On the next day, the original medium was drained, fresh medium was replaced for control group. For the OGD experimental group, cells were washed with PBS (Mlbio Co., ltd. Shanghai, China) to wash the residual medium and cell debris, and a sufficient amount of sugar-free Eagle'S solution (Gibco Life Technologies, Inc., Grand Island, NY, USA) was added and transferred to an anoxic incubator (Shanghai Yuejin Medical Instruments CO., Ltd, Shanghai, China), in which PC12 cells was lacking glucose and hypoxia for 1 h. Then the sugar-free Eagle's solution was discarded, and the complete medium was added. Cells were placed in a cell culture incubator with 5% CO2 at 37 ºC to achieve complex sugar reoxygenation. The cells were collected after reoxygenation for 24 to 48 hours.
Knockdown and overexpression of CEBPA-AS1
The corresponding overexpression sequence and shRNAs (shRNA 1: 5’-GCGCGGATTCTCTTTCAAAGC-3’, shRNA 2: 5’-GCACCGAGGGAGGAGACAAAC-3’, shRNA 3: 5’-GCGTCCCTCGCATTCTTTACC-3’) were designed according to the CEBPA-AS1 gene sequence of Genbank, and its sequence was synthesized by Wuhan Jingsai Bioengineering Technology Co., Ltd. For overexpression of CEBPA-AS1, the CEBPA-AS1 full-length lncRNA directed cloning into the multiple cloning site of the adenoviral vector. After correct identification by enzyme digestion, it was named Ad-CEBPA-AS1. The empty vector was used as control. Adenovirus overexpressing CEBPA-AS1 was injected into model rats. MiR-340-5p was simultaneously injected for rescue assay. For knockdown of CEBPA-AS1, shRNAs were transfected in OGD/R cells for 12, 24 and 48 h. The processes of transfection were followed the operation manual of Lipofectamine® 2000 (Invitrogen, Carlsbad, USA).
qRT-PCR was used to detect the expression of CEBPA-AS1 and miR-340-5p in brain tissue and cells after reperfusion. Total RNA were extracted from tissues or cells using the TRIzol reagent (Thermo Fisher Scientific, USA). 1 µg total RNA was taken as a reverse transcription template. The following PCR primers were used: CEBPA-AS1 forward sequence, 5'-CGATGTGAGCCGGAGAG-3' and reverse, 5'-TTGATTTCCGTCCAGGTCT-3'; miR-340-5p forward sequence, 5'-AGGCGCTTATAAAGCAATGAGA-3' and reverse, 5'-GTGTGGTGTGGTATGGTGTG-3'. Reverse transcription was performed according to the instructions of K1622 reverse transcription kit. After reverse transcription, fluorescent quantitative PCR was performed on a QuantStudio 3 instrument using a QuantiNovaTM SYBR® Green PCR kit (QLAGEN, Germany), and the specific procedures of the experiment followed the corresponding kit instructions.
The left MCA of the rats was sacrificed for 2 hours after reperfusion for 24 hours, and the brain was decapitated. The general morphology of the brain was observed, the cerebellum and brainstem were removed and the sample was stayed in the physiological saline (0 to 4 °C) for 10 minutes. 2 Mm thick coronal sections were placed in 2% TTC/phosphate buffer (Sigma, St Louis, MO), stayed in 37ºC for 30 minutes in the dark, and fixed in 4% paraformaldehyde/PBS for 6 hours to observe the coloration of brain infarction by Olympus microscope (Olympus, Tokyo, Japan).
Brain water content determination
The rats were sacrificed by excessive anesthesia at the corresponding time points, and about 250 g of brain tissue was taken. After weighing the wet weight of each tissue with a JA1003 electronic balance, the sample was dried in an infrared drying oven at 100 °C for 24 hours, and the dry weight was weighed. The formula for calculating brain water content was as follows: brain water content = (wet weight - dry weight) / wet weight × 100%
Nissl staining assay
The sample was routinely sliced and placed in a fume hood for 48 h. Distilled water and glacial acetic acid were used to prepare a tar purple dye solution with a concentration of 0.001 g/ml, and the solution was centrifuged at 2000 r/min for 10 min to remove the precipitated particles for use. The slices were immersed in chloroform, absolute ethanol, 95% ethanol, 70% ethanol for 1 min, soaked in distilled water for several seconds. The samples were then incubated in tar purple dye solution (Mlbio Co., ltd. Shanghai, China) at 37 °C and room temperature for 15 min, respectively. Then, the slices were washed with distilled water for 2 min, and separated by 95% alcoholic acetic acid solution for 3 times. The sections were transparent by xylene for 5 min, and sealed with neutral gum. After drying, the sample was observed under the light microscope (Jena, Germany) and photographed.
Immunofluorescence cytochemistry assay
The coverslips with cell growth for 7 days were rinsed 3 times with PBS. The cells were fixed in 40 g/L paraformaldehyde for 30 min, washed three times with PBS, 5 min/time, and blocked with 0.1% BSA for 1 h. Rabbit anti-rat NeuN antibody (Upstate, Billerica, MA) was added, the sample was incubated for 1 h at room temperature and washed with PBS for 3 times. The secondary antibody plus rhodamine labeled goat anti-rabbit IgG antibody (Abcam, UK) with an antibody concentration of 1:200 was added. After the reaction was completed, the cell-grown surface of the cover slip was placed on a glass slide in which a mixture of glycerin and PBS (1:1) was dropped. Nerve cell survival was observed by a fluorescence microscope (Oly mpus, BX 60).
Detection of lactate dehydrogenase (LDH)
The LDH activity in serum was detected by LDH kit (Clontech, Mountain View, CA, USA) following the instructions. Serum diluted in saline at a volume ratio of 1:10. Total 10 µL sample was added to the microplate. The substrate buffer and the oxidized coenzyme I solution were mixed at a volume ratio of 5:1, 60 µl of the mixed solution was added to each well. The sample was thoroughly mixed and stayed in 37 °C for 15 minutes. Add 50 µl of 2,4-dinitrophenylhydrazine solution and mix well with the sample. 37 degrees Celsius water bath for 15 minutes. 150 µl of stop reagent were added to samples and mixed well. After standing at room temperature for 3 minutes, the wavelength was measured at 440 nm.
Detection of malondialdehyde (MDA) content
The content of MDA was detected by MDA kit (Jiancheng, Nanjing, China) following the instructions. The brief steps were as follows. Cells were collected into a centrifuge tube, centrifuged, and the supernatant was discarded. The ratio of cells (104) to the volume of the extract (ml) is 500 ~ 1000:1. The cells were disrupted by sonication, centrifuged at 8000 g for 4 minutes at 4 ° C, and the supernatant was taken and placed on ice for testing. 0.3 ml of the reagent was placed in a centrifuge tube, and 0.1 ml of the sample was added and mixed. The sample was placed in a water bath for 30 min, cooled in an ice bath, and centrifuged at 25 ° C for 10 minutes. 200 µl of the supernatant was pipetted into a 96-well plate, and the absorbance at 532 nm and 600 nm was measured. The MDA content was calculated by the absorbance.
FISH assay for distribution of lncRNA CEBPA-AS1 in cells
The cell slide was placed on the bottom of a 24-well plate. A total of 5 × 103 cells were added into per well, and the supernatant was removed after 24 h of culture. Cells were washed with PBS. After being fixed with 4% paraformaldehyde, PBS containing 0.5% TritonX100 was added, and the pre-hybridization solution was used to block at 37 °C. The lncRNA CEBPA-AS1 probe was applied to hybridize overnight at 37 ° C, and the hybridization washing solution was used to wash the cells at 42 ° C. The hybridization area was stained with DAPI, and the slide was fixed in the dark. The samples were observed under a laser confocal microscope (OLYMPUS JAPAN CO., LTD, Japan).
Detection of apoptosis in nerve cells by TUNEL assay
Nerve cells were seeded in a laser confocal glass dish at 1 × 106 cells per well, and cultured in a humidity incubator for 24 h. The sample was treated by 100 ul 20 µg / ml Proteinase K for cell permeability. The sample was immersed in 4% paraformaldehyde for 5 minutes to fix. Total 100 ul of equilibration solution was added and placed in a wet box to equilibrate for 10 min. 100 µl of TUNEL reaction mixture was added to the specimen and reacted for 1 h at 37 ° C. The reaction was stopped by immersing in SSC for 15 min. After blocking and dipping, DAB was processed in the dark. The samples were counterstained with hematoxylin, dehydrated with gradient alcohol, transparent twice with xylene, and sealed with neutral gum. Finally, the sample was observed and photographed.
After paraffin sections were dewaxed and gradient alcohol dehydrated, antigen retrieval was performed. The samples were then rinsed with 0.01M PTST for 5 minutes, placed in 2% BSA and seal in a wet box at 37 ℃ for 30 minutes. Appropriately diluted fluorescently labeled antibodies were added dropwise to the samples and incubated for 30 minutes. Appropriately diluted fluorescently labeled antibodies were added dropwise to the samples and incubated for 30 minutes. Buffered glycerol is used for mounting slides. The samples were observed under a fluorescence microscope (Oly mpus, BX 60).
Transfected PC-12 cells were incubated with 5 × 103 per well in 96-well plates overnight. After culturing the cells for 48 h, 20 µL MTT (5 mg·mL− 1, Sigma-Aldrich, St. Louis, MO) was added and cultured in a 37 °C incubator for 4 h. The medium in the well was removed, 100 µL of dimethyl sulfoxide was added, and the A value was measured at 490 nm after shaking and mixing. The cell viability results were expressed as the ratio of the A values of the transfected group to the control group.
Western blot assay
Western blot method was used to detect the expression of Bcl-2, Bax, cleaved-caspase-3, cleaved-PAPR, APPL1, N-LKB1, C-LKB1, p-AMPK and AMPK. Total cellular protein was extracted. Protein separation was performed by SDS-PAGE, electric transferred to PVDF membranes (Millipore, Billerica, MA, USA)., blocked by 5% skim milk powder for 1 hour, and washed by TBS. Primary antibody was added to the blocking solution at a ratio of 1:1000 and slowly shake at 4 ° C overnight. The primary antibody was discarded, the membrane was washed by TBST, goat anti-rabbit IgG secondary antibody (Thermo Fisher Scientific) was added to membrane and stayed for 1 h. The membrane was exposure by chemiluminescence. Quantity One software was applied to analyze the data of gray scale.
Luciferase reporter system detects relative fluorescence values
The binding site of CEBPA-AS1 and miR-340-5p were determined by the online prediction software miRDB. Targetscan predicted target genes binding to miR-340-5p. To demonstrating the binding site, a mutant sequence of the CEBPA-AS1 3'UTR deletion miR-340-5p binding site and a wild-sequence insertion reporter plasmid were designed. Co-transfection of miR-340 mimics and wild-type (CEBPA-AS1-wt / miR-340-5p) or mutant (CEBPA-AS1-mut / miR-340-5p) recombination in 293T cells was processed using dual luciferase reporter assay system (Promega, Madison, WI, USA). qRT-PCR was used to detect transfection efficiency of miR-340-5p mimic. Luciferase reporter system detected relative fluorescence values. Following the same steps, the luciferase reporter assay was also used to confirm the target APPL1 of miR-340-5p.
RNA pull-down assay
CEBPA-AS1 was overexpressed in PC-12 cells, and RNA pulldown experiments were performed, followed by qRT-PCR to detect miR-340-5p enrichment. 5-bromo-UTP (BrU) labeled CEBPA-AS1, and pcDNA3.1 were synthesized. The labeled RNAs were combined with anti-BrU antibody. Extracted cytoplasmic was incubated with the RNA/antibody/beads mixture for 2 h. Afterwards, the beads were washed by Buffer II. BrdU was used to elute RNA/protein complexes and mass spectrometry analysis was processed for detection. Proteins specific binding to LncRNA CEBPA-AS1 were ranked based on spectra counts.
RNA immunoprecipitation (RIP) assay
RNA immunoprecipitation (RIP, Millipore, Bedford, MA) detected the binding site of miR-340-5p and CEBPA-AS1. The RNA lysate was used to lyse the cells. 1.5 mg of total protein per sample was taken, and 800 µl of NT-2 buffer was added. 20 mg of AG02 antibody or rabbit IgG was added at a ratio of 1:50. The washed sample was blotted dry, and then 10 µl of 2 × proteins was added, boiled at 95 ° C for 5 min, placed on ice, and subjected to Western blotting with Input for immunoprecipitation. Trizol extracted Input-RNA samples were quantified, then 2 µg of RIP-derived RNA was reverse-transcribed into cDNA, and real-time PCR was used to detect the expression of miR-340-5p and CEBPA-AS1 in the AG02 antibody complex.
Each experiment was repeated three times and data are presented as the mean ± standard error (SD). Statistical analysis was processed by SPSS17.0 (SPSS, Inc., Chicago, IL, USA). One-way ANOVA was used to determine the difference among at least three groups. The different between two groups were calculated by a t-test. P < 0.05 was regarded as significant.