Animal treatment 
A total of 75 C57/BL6 mice (aging 8–10 week old; weighing 22–25 g), were purchased from Shanghai Ruibo Biotechnology Co., Ltd.
Among the 75 mice, 60 were used to establish MCAO model, and the remaining 15 were sham-operated to serve as control. For MCAO model establishment, the mice were anesthetized with 5% pentobarbital sodium, with the right common, external (ECAs), and internal carotid arteries (ICAs) were exposed. After ligation of the common carotid arteries with sutures, a 7/0 silicone-coated monofilament nylon suture in diameter of 0.22–0.23 mm was gently introduced into the ICAs through the ECA using a rounded tip and advanced 10 ± 0.5 mm until the rounded tip reached the entrance to the right middle cerebral artery, with a slight resistance felt. Laser Doppler flowmetry (PeriFlux 5000, Järfälla, Sweden) was used to identify the successful occlusion.
Then, the 60 successfully modeled mice were randomly grouped into MCAO, MCAO + agomir negative control (NC) + overexpression (oe)-NC (agomir NC and oe-NC were stereotactically injected into the brains of MCAO mice after 1 h of modeling), MCAO + miR-140-5p agomir + oe-NC (miR-140-5p agomir and oe-NC plasmid were stereotactically injected into the brains of MCAO mice after 1 h of modeling), and MCAO + miR-140-5p agomir + oe-TLR4 (miR-140-5p agomir and oe-TLR4 plasmid were stereotactically injected into the brains of MCAO mice after 1 h of modeling) (n = 15 in each group). The stereotactic injections were employed according to a previous study . In brief, mice were anesthetized with intraperitoneal injection of 3% pentobarbital sodium. According to The Mouse Brain in Stereotaxic Coordinates: Second Edition (Deluxe) (GeorgePaxinos, Keither B J Franklin), after 1 h of MCAO, the above mentioned lentiviruses containing plasmids (2 × 108 ifu/mL, 4 µL for each plasmid) were stereotactically injected at a ratio of 1 µL/min into the mice by using a stereotaxic apparatus (KOPF, Tujunga, California, USA) and a stepper-motorized microsyringe (Hamilton, Bonaduz, Switzerland), followed by retaining the needle for 5–10 min.
Neurological function evaluation
As a previous reference reported , 6 mice in each group were randomly selected for neurological function evaluation after 24 h of MCAO. The tests included motor tests (including flexion of forelimb and hindlimb, and head movement, scored on a scale of 0–6), beam balance tests (scored on a scale of 0–6), and tests for the presence of reflexes and abnormal movements (scored on a scale of 0–2). The score was blindly evaluated by three independently researchers, with 1–4 points indicated mild injury, 5–9 points indicated moderate injury, and 10–14 points indicated severe injury.
Model identification by triphenyltetrazolium chloride (TTC) staining
Six mice in each group were euthanized after evaluating neurological function. Brains were collected and cut to 2 mm coronal sections. Each section was stained with 2% TTC solution for 30 min at 37 °C. Infarcted areas were white-stained and non-infarct zones were stained as red, which were analyzed by NIH ImageJ software.
Primary culture and treatment of neurons
The primary culture of neurons in the cerebral cortex was obtained from newborn C57BL/6 mice, which was conducted as previously reported . Lentiviral vector LV5-GFP (#25999, Addgene, Cambridge, MA, USA) used for gene overexpression, miR-140-5p mimic, and its negative control (mimic-NC) (Shanghai Genepharma, Shanghai, China). The lentivirus package was used HEK293T cells, which were then incubated in a Roswell Park Memorial Institute (RPMI)-1640 complete medium supplemented with 10% fetal bovine serum (FBS) and passed every other day. Then the lentiviruses (1 × 108 TU/mL) were added in the neurons for infection. After that, the infected neurons were induced as ischemic injury cell model by OGD treatment according to a previous report . In brief, neurons were washed by phosphate buffered saline (PBS) for three times, and added into a glucose-free Dulbecco's modified Eagles Medium (DMEM) at 37 °C. The neurons were subsequently incubated in a 5% CO2 and 95% N2 hypoxic incubator at 37 °C for 2 h, followed by incubation a 5% CO2 normoxic incubator at 37 °C.
RNA isolation and quantification
Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was employed according to the manufacturer’s instructions of TaqMan MicroRNA Assays Reverse Transcription primer (4427975, Applied Biosystems, Foster City, CA, USA). After measurement of quality and concentration using an ultraviolet spectrophotometry (Nanodrop ND1000, Thermo Scientific, Waltham, MA, USA), the isolated RNA was reversely transcribed to cDNA using PrimeScript™ RT Reagent Kit (Takara, Kusatsu, Japan). Subsequently, RT-qPCR was conducted using the SYBR Premix Ex Taq™ II (TliRNaseH Plus) kit (Takara, Kusatsu, Japan). Primers were synthesized by Shanghai Sangon Biotech (Shanghai, China), as listed in Table 1. The 2−ΔΔCT method was applied to calculate the fold changes, with U6 served as internal reference.
Primer sequences for RT-qPCR
Gene of interest
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R: 5’- GCAGGGTCCGAGGTATTC-3’
F: 5’- TGCGGGTGCTCGCTTCGCAGC-3’
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R: 5’- CCAGTGCAGGGTCCGAGGT-3’
|Note: F, forward; R: reverse.|
Protein quantification by Western blot assay
Neurons were lysed in lysis buffer containing protease and alkaline phosphatase inhibitors (C0481, Sigma-Aldrich, St. Louis, USA) at 4℃ for 30 min, followed by centrifugation at 10000 r/min for 15 min. The supernatant was collected, and the proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene fluoride (PVDF) membrane. The membrane was subsequently blocked and incubated at 4℃ overnight with diluted primary rabbit antibodies from Abcam (Cambridge, UK): rabbit anti-Bcl-2 (ab196495, 1: 1000), mouse anti-Bax (ab32503, 1: 1000), rabbit anti-cleaved caspase 3 (ab49822, 1: 500), TLR4 (ab13356, 1: 1000), NF-ҡB-p65 (ab207297, 1: 1000), and GAPDH (ab9485, 1: 2000, as internal control). After incubation, the membrane was washed with Tris Buffered saline Tween (TBST) buffer for 5 min three times, and incubated with horseradish peroxidase (HRP)-labelled goat anti-rabbit IgG secondary antibody (ab205718, Abcam, Cambridge, UK) for 1 h at room temperature. The immunoblots were visualized with enhanced chemiluminescence (ECL) reagent, and the images were captured and analyzed by ImageJ to calculate the grey value. All experiments were repeated three times.
Dual-luciferase reporter assay
TLR4 3’-UTR sequences with mutant (MUT) or wildtype (WT) miR-140-5p binding sites were constructed by Shanghai Ruibo Biotechnology Co., Ltd. The promoter sequence was cloned into the pGL-3 luciferase reporter vector. In the 24-well plate, after 24 h of cell incubation, pGL-3-TLR4 WT or pGL-3-TLR4 MUT was co-transfected with miR-140-5p mimic or mimic negative control (mimic-NC) into the HEK293T cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Fluorescence intensity was measured using a GeneCopoeia dual-luciferase reporter assay kit (D0010, Solarbio, Beijing, China) on a Promega Glomax20/20 luminometer (E5311, Shaanxi Zhongmei, Shaanxi, China). All experiments were repeated three times.
Flow cytometry for cell apoptosis
Cells ere titrated into cell suspension and seeded in a 6-well plate. After 48 h transfection, medium was aspired before the cells were washed with PBS and titrated into a single cell suspension. Then, the cells were stained with 5 µL of Annexin-V-FITC and 5 µL of propidium iodide (PI) for 15 min avoiding exposure to light. Next, the cells were re-suspended by 200 µL of 1 × binding buffer followed by cell apoptosis detection on a flow cytometry.
Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) staining
Brain tissues of mice were harvested and fixed in 4% paraformaldehyde overnight, which were then sectioned. Five randomly selected sections were dewaxed by xylene, dehydrated by gradient alcohol, and treated with 50 µL of 1% proteinase K at 37 °C for 30 min. The sections were then treated with 3% H2O2 methanol solution at 37 °C for 30 min to eliminate endogenous peroxidase activity. Following this, the sections were incubated with a prepared TUNEL working solution in a wet box at 37 °C for 1 h in dark, followed by adding with 50 µL of conversion-peroxidase solution for 30 min. After staining with diaminobenzidine and counterstaining with hematoxylin, the sections were dehydrated with gradient ethanol (50%, 70%, 90%, and 100%), cleared by xylene, and sealed with neutral balsam. Under a 40 × optical microscope, the positively stained neurons in 10 randomly selected visual fields of each section were observed as yellowish-brown, and neurons with blue nuclei were normal neurons. The neuronal apoptosis rate was calculated as the apoptosis ratio = the number of positive neurons with yellowish-brown/the number of total neurons × 100%, while the number of total neurons = neurons with yellowish-brown + neurons with blue nuclei.
Statistical data was processed by SPSS 19.0 (SPSS Inc., Chicago, IL, USA). Normal distribution and variance test were carried out, and measurement data conforming to the Normal distribution was expressed as mean ± standard deviation. Data comparisons between the two groups were analyzed by unpaired t-test, while comparisons among multiple groups were performed by one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test. A value of p < 0.05 indicated a significant difference.