Cell culture and treatment
HEK293 cell line were stored in our laboratory, while primary dorsal root ganglion (DRG) neurons were extracted from 5-week old C57BL/6 mice as previously described. Briefly, 5-week old C57BL/6 mice were anesthetized and sacrificed by cervical dislocation. L4-L5 portion of spinal cord was extracted. The dorsal root ganglia were retrieved and dissociated with 0.25% trypsin (Invitrogen, USA). The cells were washed with 2.5% bovine serum albumin (BSA, Invitrogen, USA), re-suspended in serum-free neurobasal medium (Invitrogen, USA) supplemented with penicillin/streptomycin (40,000 unit/L, Invitrogen, USA) and B-27 serum-free supplement (Invitrogen, USA). To induce neurotoxicity, the DRG neurons were treated with various concentration of bupivacaine (0.5,1.0,1.5 or 2.0 mM) for 6 h, 12 h, 24 h, 48 h.
Transfection
The knockdown vectors of lincRNA PADNA were constructed by Gene Pharma (Shanghai, China). Empty vectors and vectors with the wild-type (WT) or mutant (mut) binding sites for miR-194 were constructed by Gene Pharma (Shanghai, China). The 3′-untranslated region (UTR) of FBXW7, with wild-type (WT) or mutant (mut) binding sites for miR-194, was amplified and cloned into the pGL3 vector (Promega, Madison, WI) to generate the vector pGL3-WT-FBXW7-3′-UTR or pGL3-mut-FBXW7-3′-UTR. The miR-194 mimic, miR-194 inhibitor, mimic NC, inhibitor NC were purchased from Shanghai Gene Pharma (Shanghai, China). Briefly, the DRG neurons were transfected with vectors, miR-194 mimic, miR-194 inhibitor, mimic NC and inhibitor NC by lipo3000 reagent (Invitrogen) according to the manufacturer’s protocol. Cells were incubated for 48 h before further researches.
Cell Viability Assay
The DRG neurons were seeded into 96-well plates and treated with bupivacaine (0.5,1.0,1.5 or 2.0 mM) for 24 h to establish bupivacaine-induced neurotoxicity model. Each well was added 10 µL 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) solution(5 mg/mL, Beyotime, Shanghai, China) and incubated for 4 hours at 37 °C. Cell viability was measured by a microplate reader at 570 nm (Bio‐Tek, Winooski, VT).
Caspase-3 Activity
A caspase-3 activity assay kit (Beyotime, Shanghai, China) was used to assess caspase‐3 activity according to the manufacturer’s protocol. DRG neurons were lysed and centrifuging. A final concentration of 0.2 mM ADEVD-pNA (caspase-3 substrate) were added into the cell supernatant and incubated for one hour. The caspase-3 activity was measured by a microplate reader at 405 nm (Bio‐Tek, Winooski, VT).
Tunel Assay
A terminal deoxynucleotidy1 transferase- mediated dUTP nick end-labeling (TUNEL) assay were conducted to evaluate the apoptosis of DRG neurons. DRG neurons were incubated with TdT and fluorescein-labelled dUTP for 45 min at 37 °C. Then FACSCalibur flow cytometer was used to measure the percentage of apoptotic of cells.
Dual-luciferase Reporter Assay
Empty vectors or vectors with the wild-type(WT) or mutant (mut) binding sites for miR-194 were co-transfected with miR-194 mimic or mimic NC. Luciferase activity was analyzed using dual-luciferase reporter system following the manufacture’s protocol. Firefly luciferase activity and Rinilla luciferase activity were measured with Multiskan Spectrum (Thermo Fisher, USA). Similarly, empty vectors or vectors of 3’-UTR of FBXW7 with their wild-type or mutant binding sites for miR-194 were co-transfected with miR-194 mimic or mimic NC. Luciferase activity was analyzed using dual-luciferase reporter system. Firefly luciferase activity and Rinilla luciferase activity were measured with Multiskan Spectrum (Thermo Fisher, USA).
Real-time Pcr
Total RNA was extracted by TRIzol reagent (Thermo Fisher Scientific). RNA reverse transcription was performed using a PrimeScript™ RT reagent Kit with gDNA eraser (RR047A; Takara, Tokyo, Japan) and cDNA was performed using SYBR® Premix Ex Taq™ (RR420A; Takara, Tokyo, Japan). The data were normalized using β-actin levels and further analyzed by the 2 − ΔΔCT method.
Western Blotting
DRG neurons were harvested and lysed by RIPA lysis buffer containing proteinase inhibitor (Roche, USA). Total protein was quantified using BCA protein assay kit (Pierce, Rockford, IL, USA). Protein samples were resolved by 10% SDS-PAGE gel and transferred to polyvinylidene difluoride membrane. After blocked, they were incubated with primary antibodies against caspase3 (1:1000, Abcam, MA, USA ), FBXW7 (1:1000, Abcam, MA, USA) and Actin (1:1000, Abcam, MA, USA) at 4 °C overnight, followed by incubation with a peroxidase-conjugated goat anti-rabbit (or mouse) IgG antibody. Immunopositively bands were analyzed using a FluorChem M system (ProteinSimple, San Jose, CA, USA).
Data analysis
We used SPSS 23.0 to calculate the values (means ± standard error of the mean). Statistical analyses were analyzed using two-sided Student’s t-test or one-way ANOVA. The statistical significance was P < 0.05.