Animals
Male Balb/c mice aged 8 weeks were purchased from Animal Experimental Center, Sun Yat-sen University, China (Guangzhou, China). Mice were housed in separated cages and the rooms were kept at 24 ± 1℃ (297 ± 1 K) temperature and 50–60% humidity, under a 12/12 light-dark cycle and with free access to food and water ad libitum. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Guangzhou Medical University and were carried out in accordance with the guidelines of the National Institutes of Health on animal care and the ethical guidelines for investigation of experimental pain in conscious animal.
Cell Culture And Transfection
Culture and transfection of BV2 cells were carried out as described elsewhere [22]. All cells were collected in high glucose Medium (Gibco) with 10% FBS (JRScientific, Woodland, CA, USA), 100 mg/mL streptomycin, and 100 units/mL penicillin (Quality Biological, Gaithersburg, MD, USA). The cells were incubated at 37 C, 95% O2, and 5% CO2 with a seeding density of 1*105 BV2 cells/mL. One day later, miRNAs mimic or inhibitor (RiboBio, Guangzhou, China) were transfected in the cells. 120 ul Opti-Mem was used to dilute miR-106b-5p mimic (50 nM)/inhibitor (100 nM) or negative control. The sequences for miR-106b-5p mimic are as follow, forward: 5-UAAAGUGCUGACAGUGCAGAU-3, Reverse: 5-AUUUCACGACUGUCACGUCUA-3; the sequences for miR-106b-5p inhibitor is 5-AUUUCACGACUGUCACGUCUA-3. The final concentrations of mimic and inhibitor were 50 nM and 100 nM, respectively. 120 ul Opti-Mem was simultaneously used to dilute 7.5 uL RNAiMAX (Invitrogen, Carlsbad, CA), then the two solutions were mixed. After 5 min, the mixture was placed into each 2 mL well and high glucose medium was added. 2 ml LPS (100 ng/mL, Sigma) was added to each 2 mL well 6 h before the the cells were collected 48 h later for qPCR and western-blot examinations.
Cell Proliferation Assay
Cell proliferation was evaluated using a cell counting kit-8 (CCK-8) assay (Beyotime, China). BV2 cells (5 × 103) were plated onto 96-well plates, then the LPS or miRNA mimic or miRNA inhibitor were added in the BV2 cells. Each well contained 100 µL of medium supplemented with 10 µL of CCK8 solution. The absorbance at 450 nm was measured to assess the degree of cell proliferation after incubating the cells at 37 °C for 1 hr.
Luciferase Assay
A dual luciferase reporter assay was performed as outlined for a previous procedure [23]. The pmirGLO dual-luciferase vector (pmirGLO vector), which contained both the firefly luciferase gene and the renilla luciferase gene, was purchased from Promega (Madison, WI, USA). P2X4R 3’UTR, including the predicted binding sites of miR-106b-5p, was inserted into the 3’UTR region downstream of the firefly luciferase gene of the pmirGLO vector (pmir-GLO-UTR). HEK293 cells were cultivated in high glucose medium (Gibco) with 10% FBS (JRScientific, Woodland, CA, USA), 100 mg/mL streptomycin, and 100 units/mL penicillin (Quality Biological, Gaithersburg, MD, USA). The cells were incubated in a humidified incubator with 5% CO2 at 37℃. When the HEK293T cells had a confluency of 70–80%, A P2X4R-luciferase-reporter construct (pmiRGLO-P2X4R 3′-UTR vector), containing the miR-106b-5p binding motif, was co-transfected with miR-106b-5p mimic at different doses of 10, 20, and 50 nM (20 umol/L) or NC mimic into HEK293 cells by using RNAiMAX (Invitrogen). After another 48 h of culture, we used 1 * passive lysis buffer to lyse the transfected cells, and 20 ul supernatant was achieved for luciferase activity using the Dual-Luciferase Reporter Assay System (Promega). The ratio of firefly activity to renilla activity was recognized as relative reporter activity. Experiments were performed in triplicate and repeated three times.
Surgical Procedures And Drug Infusion
SNI surgeries were performed to mice according to our previous work [24]. In brief, mice were deeply anesthetized after the behavioral tests. The skin of the lateral thigh was incised and the biceps femoris muscle was dissected bluntly to expose the left sciatic nerve and its three terminal branches (the sural, common peroneal and tibial nerves). The common peroneal and the tibial nerves were tightly ligated with 5 − 0 silk. Then, the nerve was transected distal to the ligation, and 2–4 mm length of nerve fiber was removed. Great care was taken to avoid any contact with or stretching of the intact sural nerve. The wound was closed in two layers. The sham group was given all procedures except ligation and transection.
The method of intrathecal injections was described previously [25]. In brief, mice were restrained with the left hand and the injection was performed with the right hand. The vertebral landmarks for L5 and L6 vertebrate were identified by palpation. An injection into the subarachnoid space between the L5 and the L6 vertebrae was done via a 27-gage needle. Entry of the needle was confirmed with the presence of a tail flick. The injection volume of all other compounds was 5 µl. In on part, the mice were divided into three groups: sham + scramble (saline), SNI + scramble (saline) and SNI + miR-106b-5p agomir (a selective mimic of miR-106b-5p). In another part, the mice were divided into two groups, naïve + scramble and naïve + miR-106b-5p antagomir. Beginning 1 day after naïve or 7 days after SNI surgery, continuous intrathecal infusion was delivered once a day for 3 days, from day 1 to 3 for naïve mice and from day 7 to 9 for SNI mice. Mice with neurological deficits were excluded from the experiment.
Behavior Tests
The 50% paw withdrawal thresholds (PWMTs) were measured on days 0, 3, 5, and 7 for the evaluation of SNI model or on days 0, 5, 7, 8, 9, 10, and 14 during continuous miR-106b-5p agomir injection following SNI surgery. They were measured on days 0, 1, 2, and 3 during continuous miR-106b-5p antagomir injection in naïve mice, always between 8 and 10 AM in the morning. PWMT was assessed with method described previously [26]. The filament force evoking paw withdrawals for more than 3 times in a round of testing was defined as the mechanical threshold. The cutoff force was 4 g. The observation of a positive response (paw lifting, shaking, or licking) within 5 s.
Mirna Microarray Analysis
The method was described as our previous study [13]. MiRNA expression profiling was performed using the RiboArray platform (RiboBio, Guangzhou, China). In brief, 1 lg of total RNA was labeled with a Cy3 using a ULS™ microRNA Labeling Kit (Kreatech, Amsterdam, Netherlands) and hybridized on the microarray. Based on Sanger miRBase version 19.0 database, RiboBio designed 1263 specific oligos for 1281 mouse miRNA, where 1263 are non-redundant sequence. In addition, 54 RiboArray™ internal controls were used. We also put some probes for location identify function. T-test P-value of < 0.05 and fold-change (> 2) were applied to determine two differentially expressed (DE) sets of genes of six experimental samples.
Rna Extraction And Quantitative Real Time Pcr (qpcr)
Total RNA was extracted using the Trizol method [24, 27]. The quality and quantity of RNA were measured using a Nanodrop Spectrophotometer (Thermo Scientific) and samples with an absorbance ratio at 260/280 between 1.8–2.2 were considered acceptable. RNA degradation was not assessed. RNA dilutions were made up in nuclease-free water. For analysis of mRNA, the method was described previously [24, 27]. Reverse transcription was generated using the SuperScriptTM III Reverse Transcriptase (Invitrogen) with a Gene Amp PCR System 9700 (Applied Biosystems). For miRNA analysis, 1 ug RNA was used for reverse transcription by miRNA 1st Strand cDNA Synthesis Kit (by stem-loop) (Vazyme, #MR101) according to the instructions. Quantitative Real-time PCR (qPCR) was carried out on a real-time detection instrument ViiA 7 Real-time PCR System (Applied Biosystems) using 2X PCR master mix (Arraystar). All experiments were replicated three times. The relative expression of genes was calculated based on the 2−△△Ct method using the mouse housekeeping GAPDH/U6 gene as an endogenous control. Expression ratios were subjected to a log2 transform to produce fold change data.
Western Blot
To ensure a sufficient amount of protein, BV2 cells and a section of ipsilateral Lumbar enlargement was prepared. Based on established protocol [28], tissues were homogenized in a RIPA buffer (Sangon, Shanghai, China). After centrifugation at 12 000 × g for 15 min at 4 °C, the supernatant was collected to analyze cytosolic proteins, and protein concentrations were determined with a BCA Kit (Thermo Fisher Scientific, Rockford, IL, USA). The contents of the proteins in the samples were measured using the Bio-Rad protein assay (Bio-Rad) and were then heated at 99 °C for 5 min. Samples of 20 mg total protein were separated by 10% SDS-polyacrylamide gel electrophoresis and electrophoretically transferred onto a polyvinylidene difluoride membrane. After the membranes were blocked with 5% Milk in Tris-buffered PBS containing 0.1% Tween-20 for 1 h, then rabbit anti-P2X4R (1:1000, Alomone Labs), rabbit anti-tubulin (1:10000, Sigma), and rabbit anti-GAPDH (1:5000, Cell Signaling Technology) primary antibodies would be used. The proteins were detected using horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:1000, Jackson) and visualized using Western peroxide reagent and luminol/enhancer reagent (Clarity Western ECL Substrate, Bio-Rad). The intensity of the blots was quantified via densitometry using Image J software. All cytosol protein bands were normalized to tubulin or GAPDH.
Fluorescence In Situ Hybridization
The fluorescence in situ hybridization technology was performed by Servicebio (wuhan, China). The spinal cord of animals was sectioned at 3 mm, and sections were dehydrated in series of ethanol washes (70%, 85%, and 100%) and air-dried. After incubated in hybridization solution at 37 °C for 1 h, the sections were incubated overnight in hybridization solution with 6 ng/µL of DIG (488) labeled probes for miR-106b-5p (5’-DIG-ATCTGCACTGTCAGCACTTTA-DIG-3’) at 37 °C. Following hybridization, the sections were washed twice at 37 °C with 2 × SSC for 10 min, then twice at 37 °C with 1 × SSC for 5 min and in 0.5 × SSC at room temperature for 10 min. After they were blocked for 30 min at room temperature, we incubated them with anti-DIG-HRP at 37 °C for 50 min. Finally, FITC-TSA was added at dark for 5 min. To identify the cell types expressing miR-106b-5p, the above sections were incubated overnight at 4 °C with primary antibodies against ATP-gated cation channel protein (P2X4R, rabbit, 1:100, ), the ionized calcium-binding adapter molecule (IBA-1, rabbit, 1:300;), glial fibrillary acidic protein (GFAP, mouse, 1:1000;) or the neuronal-specific nuclear protein (NeuN, mouse, 1:50;) for double staining as described previously.
Statistical analysis
The data are presented as means ± SEM. For comparisons between two groups, the P value was evaluated and calculated using a two-tailed unpaired t-test. When there were multiple groups involved, a one-way analysis of variance (ANOVA) was used; multiple factors were compared using two-way analysis of variance (ANOVA). Values of P < 0.05 were considered statistically significant.