Animals
Spargue-Dawley male rats (220-260 g) were obtained from the Institute of Experimental Animals in Sun Yat-Sen University. The animals were kept at room temperature with 50 % to 60 % humidity, and were housed in the separated cages with ad libitum access to food and water. All experiment procedures were approved by the Local Animal Care Committee and were performed in accordance with the guidelines of the National Institutes of Health on animal care and the ethical guidelines.
Drug administration and Behavioral test
Solution of paclitaxel was prepared before each application and injected intraperitoneally (8 mg/kg/day) on day 1, 4 and 7. The withdrawal threshold of foot was determined by applying mechanical stimuli to the plantar surface of the hindpaw using Von Frey hairs, and 50% withdrawal threshold was determined using the up-down method [16, 21]. NFATc2 siRNA (50 μg/15μl, Ribobio), CXCL14 siRNA (50 μg/15μl, Ribobio), FK506 (10 μg/10μl) or scrambled siRNA was intrathecally injected for consecutive 10 days.
Intrathecal injection was preformed according to our previously described method [22, 23]. In brief, polyethylene catheters (PE-10) was inserted into the lumbar subarachnoid space between 5th and 6th lumbar vertebrae with the tip of the catheter located near the L5 spinal segmental level. Following intrathecal implantation of catheters, animals were allowed 5 days to recover from surgery prior to subsequent drug injection. Animals, which exhibited hind limb paresis or paralysis after surgical operation, were excluded from the experiment.
Immunohistochemistry
Perfusion was performed through the ascending aorta with 4% paraformaldehyde after an application of sodium pentobarbital at 50 mg/kg dose (i.p.). The lumbar segments of the spinal cord were removed and placed into 4% paraformaldehyde for 3 h, and stored in 30% sucrose overnight. Cryostat sections (16 µm) were cut and processed for immunohistochemistry with primary antibodies for CXCL14 (1:200; Abcam), NFATc2 (1:200, Thermo Fisher Scientific), Iba1 (1:25, Sigma), NeuN (1:200, Chemicon) and GFAP (1:200, Chemicon). After incubation overnight at 4℃, the sections were incubated with secondary antibodies, which conjugated with cy3 or fluorescein isothiocyanate for 2 hours at RT. The stained sections were then examined with a Leica (Leica, Germany) fluorescence microscope, and images were captured with a Leica DFC350 FX camera.
Western blot
Animals were anesthetized with sodium pentobarbital (50mg/kg, i.p.) at various time points. The spinal cord was removed and sectioned in a cryostat. Dorsal horn tissue was punched with a 15-gauge cannula and homogenized in 15mmol/l Tris containing a cocktail of proteinase inhibitors and phosphatase inhibitors on ice. Protein samples were separated by gel electrophoresis (SDS-PAGE) and transferred onto a PVDF membrane. The membrane were placed in the blocking buffer for 1 h at room temperature and incubated with primary antibodies against CXCL14 (1:1000; Abcam), NFATc2 (1:1000, Santa Cruz), acetylated histone H3 (1: 1000, Millipore), acetylated histone H4 (1: 1000, Millipore) or β-actin (1:2000, Cell Signaling Technology) overnight at 4℃. Then, the blots were then incubated with horseradish peroxidase-conjugated secondary antibody for 2 hours at RT. ECL (Pierce, USA) was used to detect the immune complex. The bands were quantified with a computer assisted imaging analysis system (NIH ImageJ).
siRNA preparation and screening
Specific siRNAs were used to knockdown the expression of CXCL14 and NFATc2. Three siRNA targeting rat CXCL14 gene or NFATc2 gene were designed and synthesized by Ribobio (Guangzhou, China) for the subsequent experiments, respectively. The siRNA sequence of CXCL14 gene and NFATc2 gene were shown in Supplemental Table 1. According to the previous screening test, siRNAs were transfected into the HBZY-1 cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). The siRNA, which has no homology to CXCL14 or NFZTc2 gene, was used as control (Scramble). The expression of CXCL14 mRNA was suppressed by 87.77±6.19%, 33.92±7.56%, 59.90±6.08% in cell lines when treated with CXCL14 siRNA1, 2, 3. The expression of NFATc2 mRNA was suppressed by 87.85±7.53%, 57.84±8.33%, 28.05.±9.65% in cell lines when treated with NFATc2 siRNA1, 2, 3.Consistent with the suppression of mRNA, the CXCL14 and NFATc2 protein level were remarkably reduced after CXCL14 siRNA1 or NFATc2 siRNA1 treatment (Supplemental Figure. 1). Therefore, the chemically synthesized CXCL14 siRNA1 and NFATc2 siRNA1 were chosen for the subsequent experiments in vivo.
RNA extraction and real-time qPCR
Trizol was used to extract total RNA from dorsal horn tissues. The reverse transcription was performed according to the manufacturer’s protocol of polymerase chain reaction (PCR) production kit. the primers sequences for PCR assay on all targeted mRNAs were presented in Supplemental Table 2. Real-time qPCR was performed using SYBR Green qPCR SuperMix (Invitrogen) and the ABI PRISM7500 Sequence Detection System. The reactions conditions included incubation at 95 °C for 3 minutes followed by 40 cycles of thermal cycling (10 s at 95°C, 20 s at 58°C, and 10 s at 72°C). The ratio of mRNA expression was analyzed by the 2-ΔΔCT Method.
Coimmunoprecipitation (Co-IP)
Coimmunoprecipitation was carried out using the Coimmunoprecipitation Kit (Pierce, Rockford, IL). Briefly, spinal dorsal horn tissues were excised quickly and put into lysis buffer. The p300 antibody or NFATc2 antibody, which was immobilized with resin, was used to collect the immune complexes. The eluted complexes from the resin were analyzed by Western blot using NFATc2 antibody or p300 antibody after incubation and washes.
Chromatin Immunoprecipitation (ChIP)
ChIP assays were performed using the ChIP Assay Kit (Thermo) as described previously [17]. The animal’s L4 and L5 spinal cord was removed quickly and placed in 1% formaldehyde for 10 min at room temperature to cross-link transcription factors with chromatin. The formaldehyde was then inactivated by addition of 125 mM glycine. Sonicated chromatin extracts containing DNA fragments were immunoprecipitated using 6 μg of ChIP-grade NFATc2 antibody or normal rabbit IgG antibody with pre-blocked protein G-Sepharose beads overnight at 4 ℃. The next day, the chromatin-protein-antibody-bead complexes were eluted and the DNA was extracted. The precipitated DNA was resuspended in the nuclear-free water, and qPCR assays with primers (Supplemental Table 3) were performed to amplify the different region within the CXCL4 promoter, containing the NFATc2 motif (red font). Finally, the ratio of ChIP/input in the spinal dorsal horn was calculated.
ChIP-seq identification of NFATc2 binding sites
ChIP-seq libraries were prepared from a total of 10 ng DNA using TruSeq Nano DNA Sample Prep Kit (Illumina) according to the manufacturer’s instructions. The completed libraries were quantified by 2100 Bioanalyzer (Agilent, Waldbronn, Germany). The libraries were then sequenced by running 2×150 cycles on the Illumina HiSeq 4000 following the HiSeq 3000/4000 SBS Kit protocol (Illumina). After the sequencing platform generated the sequencing images, the stages of image analysis and base calling were performed using Off-Line Basecaller software V1.8. Sequence quality was examined using the FastQC software. After passing Solexa CHASTITY quality filter, the clean reads were aligned to Rat genome (UCSC RN5) using BOWTIE software V2.1.0 [24]. The MACS V1.4.2 program [25] was then used for peak calling of the ChIP enrichment regions relative to control data set that was generated from input samples. The peaks in samples were annotated by the nearest gene using the newest UCSC RefSeq database.
Microarray analysis
Total RNAs were reverse transcribed into double-stranded cDNAs. Then cDNAs in-vitro transcribed into antisense cRNAs and labeled with Cy3-CTP and Cy5-CTP using a Two-Color Low Input Quick Amp Labeling Kit (Agilent Technologies, Santa Clara, CA, USA). Fluorescence dye labeled cRNAs were fragmented and hybridized on Sure Print G3 Rat GE 8x60K Microarray using an Agilent Gene Expression Hybridization Kit. The fluorescence intensities at 635 nm (Cy5) and 532 nm (Cy3) were scanned by an Agilent microarray scanner. Microarray data were extracted using Agilent Feature Extraction Software. Low intensity spots were removed, and each gene had expression value in more than 80% samples analyzed. Signals were normalized by Loess normalization. Differentially expressed genes (DEGs) were screened using SAM v4.01 software, with the false discovery rate set to 5% (q-value <0.05) [26].
Statistical analysis
All data were expressed as the means ± SEM and analyzed with SPSS 22.0 (SPSS, USA). Western blot and qPCR data were analyzed with two-way analysis of variance (ANOVA) followed by a Tukey post-hoc test. For behavioral tests, one-way or two-way ANOVA with repeated measures followed by a Tukey post-hoc test was carried out. The criterion for statistical significance was P < 0.05. While no power analysis was performed, the sample size was based on previous studies of painful behavior and pertinent molecular studies.