Genome Mapping, KEGG Pathways, and ncRNA Regulatory Network Analysis
The sequencing data of SCI tissues were derived from previous studies , The samples were divided into an SCI group and a Sham group, with three replicates each. The tissue samples were derived from the spinal lesion epicenter of modified standard Allen’s drop mouse model, 3 days after SCI. Sample reads were aligned to the reference genome from Ensembl or National Center for Biotechnology Information databases using the Tophat 2  package, which initially removed a portion of the reads based on the quality control information accompanying each read and then mapped the reads to the reference genome. Tophat allows multiple alignments per read, builds a database of potential splice junctions, and compares the previously unmapped reads against a database of putative junctions. The aligned read files were processed by in-house scripts, which use the normalized RNA-seq fragment counts to measure the relative abundances of the transcripts. The unit of measurement is fragment per kilobase of exon model per million (FPKM) reads mapped. CIRCExplorer was used for denovo assembly of the mapped reads to circRNAs . The assembled circRNAs from all samples were used to identify unique circRNAs; circRNAs were identified if they had a statistical p-value of < 0.05.
For pathway significance-enrichment analysis, we used a major public database, KEGG (https://www.kegg.jp/) . To determine the most important biochemical, metabolic, and signal transduction pathways, a hypergeometric test was applied to isolate the significantly enriched pathways involving genes that were differentially expressed, compared with the whole-genome background.
An ncRNA regulatory network was constructed to profile the interactions and functional links among dysregulated messenger RNAs (mRNAs), miRNAs, and circRNAs in the acute stage of SCI. The targets of miRNAs were predicted by TargetScan (http://www.targetscan.org/) by adopting the default parameters, as previously described . CircRNA/miRNA/mRNA interaction networks were constructed using Cytoscape (San Diego, CA, USA).
Construction of mouse SCI model
Eighteen (nine for the SCI group and nine for the Sham group), healthy, male C57BL/6 mice were used for our animal models, following a previously used approach . In brief, in the SCI group, the exposed spinal cord was struck using a 6 g weight, which was dropped from a height of 6 cm (Allen’s drop). The sham group had the T8–T10 segments of the thoracic vertebra exposed without an Allen's drop. At each experimental time point (3, 7, and 14 days after surgery), three mice from each group were randomly selected. Tissue was then extracted for quantitative reverse-transcription PCR (qRT-PCR) and Western Blot.
Primary cell extraction, Culture, Cytological identification and Transfection
Tissue from twenty C57BL/6 mice was used for primary spinal fibroblast extraction within 3 days of birth. After the isolated spinal cord was cut into pieces, 0.125% trypsin of 10 times volume was added and digested for 15 min in a magnetic mixer at 37℃. Medium was added for neutralization and the mixture was centrifuged at 1,000rpm for 5 min; then, the supernatant was discarded. The pellet was cultured in t-25 culture bottles, placed in an incubator at 37℃ with 5% CO2, and cultured in modified Eagle's medium (Life Technologies, Carlsbad, CA, USA) containing 10 % fetal bovine serum (Gibco, Brisbane, Australia) and 100 IU/mL penicillin–streptomycin (Gibco). The medium was renewed after 24 hours. The cell density reached about 90% after a week of continuous cell culture. The cells were digested normally with 0.25% trypsin, placed in an incubator for 30-40 min after placing the culture flasks back inside the incubator, the culture medium was aspirated, and the non-adhered cells were discarded. Newly made culture medium was added and the cells were placed in an incubator at 37℃ with 5% CO2. The medium was changed 2-3 days later. Fibroblasts were identified by immunofluorescence, as previously described . In brief, after washing, permeating, and blocking, cells were incubated with Type I collagen antibody (Abcam, Cambridge, UK) at 4℃, overnight. After incubating with a secondary antibody (Invitrogen, CA, USA) and staining with 4',6-diamidino-2-phenylindole (DAPI; Invitrogen), immunofluorescence was analyzed under a fluorescence microscope (Olympus Corporation, Tokyo, Japan). Transfection of siRNA-CDR1as (siB2009231003539885, RiboBio, Guangzhou, China, GCCGTATCCAGGGTTTCCA), miR-7a-5p mimics, miR-7a-5p inhibitor, and their negative controls (RiboBio) was initiated when the cells were 30%–50% confluent. After 48 hours, the cells were stimulated with TGF-β (10 ng/ml, ACRO, Beijing, China) for 48 h.
RNA preparation and PCR validation
All qRT-PCR assays were carried out as previously described . In brief, total RNA was isolated and then verified/quantified by the OD260/280 absorbance ratio, then reverse-transcribed into cDNA. qRT-PCR was performed using the Applied Biosystems (Wilmington, DE, USA) 7500 RT-PCR system and repeated three times. GAPDH was used as an internal control to normalize relative circRNA and mRNA expression levels. miRNA expression levels were normalized by U6. The 2–ΔΔCT method was used for comparative quantitation. The specific primers used for each target were as follows: mmu-CDR1as-Forward: AATCTATGCCTTCCACAAATG; mmu-CDR1as-Reverse: GACCTTGACAGTGTTGGA; mmu-miR-7a-5p-Forward: TGGCGGTGGAAGACTAGTGAT; mmu-miR-7a-5p Reverse: TGTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACACAACA; TGF-βR2-Forward: CATCGCTCATCTCCACAGT; TGF-βR2-Reverse: ACACAGGCAACAGGTCAA; GAPDH-Forward: GTGGTGAAGCAGGCATCT; GAPDH-Reverse: GGTGGAAGAGTGGGAGTTG; U6-Forward: CTCGCTTCGGCAGCACA; U6-Reverse: AACGCTTCACGAATTTGCGT.
Reverse-transcriptase polymerase chain reactions (RT-PCR) were carried out in accordance with according to the manufacturer's instructions found in the Select RT kit (RiboBio, Guangzhou, China) The cDNA amplification reactions were performed with 2× Taq plus RTase Mix (RiboBio) and RT primer(C)*(10μM). PCR products were separated by electrophoresis on an agarose gel (1.5%) at 120 V of constant pressure for 30 min. Finally, Sanger sequencing (m-CDR1as-DP-219bp-R1) was performed. The specific primers for Sanger sequencing and RT-PCR were as follows: mmu-CDR1as-DP-219bp-Forward 1: GTGTATCGGCGTTTTGACATTC; mmu-CDR1as-DP-219bp-Reverse 1: ACTGAATTTACTGGAAGACTCTGAG; mmu-CDR1as-CP-201bp-Forward 2: TCCACATCTTCCCAAAATCCA; mmu-CDR1as-CP-201bp-Reverse 2: GAATGTCAAAACGCCGATACAC.
Protein isolation and Western blot
Total proteins were extracted then separated by electrophoresis on sodium dodecyl sulphate-polyacrylamide gels (Solarbio, Beijing, China) and subsequently transferred to 0.22-mm polyvinylidene difluoride membranes (Solarbio). The membranes were blocked with 5% skim milk at room temperature for 1 hour. Then, the membranes were incubated with primary antibodies overnight at 4℃. Incubation with secondary antibodies (Solarbio) was performed the following day for 2 hours at room temperature. The antibodies (anti-Fibronectin, anti-Smad7, anti-p-Smad2, anti-p-Smad3, anti-Smad2/3, and anti-GAPDH antibody) used in this study were formulated as previously described [20, 21]. The protein levels were visualized using the West Pico ECL Substrate (Solarbio), and GAPDH was used as an endogenous control for normalization.
Luciferase reporter assay and fluorescence in situ hybridization
293T cells were inoculated into 96-well plates at a confluence of 70%. The plasmids pMIR-REPORT-CDR1as (WT) (LC Science, Houston, TX) and pMIR-REPORT-CDR1as (MT1+MT2) (LC Science) were transfected 24 hours later. 0.2g concentration of transfection reagent was used, the final concentration of microRNA was 100 nM. The DNA, miRNA, transfection reagent, and incubate were diluted and set at room temperature for 5 minutes. The diluted DNA and miRNA were mixed with the transfection reagent (Invitrogen), respectively, and incubated at room temperature for 20 minutes. Approximately 50 μL of culture medium was discarded from each well and 25 μL DNA transfection mixture and 25μL miRNA transfection mixture were added to each well. Data were measured after 2S. Following the addition of 50μL of pre-mixed Stop&Glo reagent to each well. Fluorescence was detected using the Opera Phenix HCS system (PerkinElmer, MA, USA).
Statistical analyses were carried out using GraphPad Prism software, and data are expressed as mean ± standard deviation (SD). Statistical significance between the two groups was assessed using independent-samples t-test, while analysis of variance (ANOVA) with post-hoc Dunnett’s corrections was performed for comparison between two or more groups. P < 0.05 was considered statistically significant.