2.1. Sample collection and cell culture
The specimens of the posterior longitudinal ligament were provided by patients from the First Affiliated Hospital of Harbin Medical University. Every patient underwent computed tomography (CT) examination, and OPLL was diagnosed if the thickness of the ectopic OPLL was more than 2 mm [7]. We set patients with only cervical disc herniation as a control group. In all 6 patients, we used Anterior Cervical Discectomy and Fusion (ACDF) surgery. During the operation, the ligament tissue (OPLL tis/sue n=3, normal PLL tissue n=3) was obtained while spinal cord decompressing. (Figure 1. AB) The surgically resected tissue was stained with HE and Masson to confirm that the collected tissue was posterior longitudinal ligament tissue. (Figure 1. CDEF)
The study protocol was approved by the Institutional Review Broad of Harbin Medical University, Harbin, Heilongjiang Province, China. Informed consent was acquired from all study participants. All research methods are within relevant ethical principles.
Primary cell culture of the posterior longitudinal ligament: The tissue obtained during surgery was rinsed with PBS. Removal of ossified tissue under the microscope and cutting into 3 pieces about 0.5mm in size, after rinsing with PBS, pieces were inoculated in T25 culture flasks containing high glucose DMEM culture medium (20% FBS, 100U/ml penicillin, 0.1mg/ml streptomycin) in a 37°C, 5% CO2, saturated humidity incubator for static culture. After 3-6 hours, the tissue blocks adhered to the wall, turning the culture flask over and immersing the tissue block in a DMEM medium. The Medium was changed twice a week and cell morphology was observed in the electron microscope. When a mass of cells sprouted around the tissue block, they were digested and passaged with 0.25% trypsin containing EDTA. Cells from the second passage were used in downstream experiments.
2.2 Sequencing of circRNAs and Microarray analysis
The purity and concentration of total RNA samples were determined with NanoDrop ND-1000. The total RNA of each sample was treated with RNase R to enrich circular RNA. The enriched circular RNA was amplified and transcribed into fluorescent cRNA utilizing random primer. After hybridizing, incubating and washing, raw data was extracted by Agilent Feature Extraction software.[18]
A series of data processing including quantile normalization were performed using the R software limma package. The circRNAs that at least 3 out of 6 samples have flags in "P" or "M" (defined by GeneSpring software) were retained for further differential analyses. [19]The microarray data have been uploaded in the National Center for Biotechnology Information Gene Expression Omnibus (GEO, https://www.ncbi.nlm.nih.gov/geo/) [GSE164546: GEO].
2.3 Differential expression analysis
Comparing two groups of profiles, the fold change (FC, the ratio of the group average) between each circRNA was calculated. The statistical significance of the difference may be conveniently estimated with the t-test. CircRNA with FC ≥ 2.0 and p-value ≤ 0.05 was selected as the significantly differentially expressed. Microsoft Excel’s Data/Sort & Filter functionalities were used to filter the analysis output, and the differentially expressed circRNA was sorted according to the fold change, p-value, etc.
2.4 Annotation for CircRNAs binding site
TargetScan and miRanda's miRNA target prediction software were used to predict the circRNA-microRNA interaction, and the differentially expressed circRNAs within all the comparisons were annotated in detail with the circRNA-miRNA interaction information. The miRNA response elements (MRE) predicted by the two algorithms were retained. We predicted the top 5 miRNAs of each circRNA based on the pairing scores, and constructed a miRNA-circRNA network through cytoscape_v3.8.0 software (http://cytoscape.org/).
2.5 GO annotation and KEGG pathway enrichment analyses of the DEGs
Gene Ontology (GO), including biological processes (BPs), cellular components (CCs), molecular functions (MFs), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enrichment analyses of DEGs were performed using the Database for Annotation, Visualization, and Integrated Discovery (P<0.05 as the criteria for enrichment significance).[20]
2.6 Protein-Protein Interaction (PPI) Network construction
To further explore the interaction of different target genes and the molecular mechanism of OPLL, we used STRING (https://string-db.org/) to analyze and construct a PPI network. Then the interaction network and the top 50 hub genes were visualized by cytoHubba in Cytoscape. The nodes in the network were represented as target genes, and the lines between the two nodes were denoted interactions.
2.7 Reverse transcription and quantitative PCR (RT-qPCR)
Total RNA were extracted from OPLL tissues using Trizol reagent (Invitrogen, CA), and a reverse-transcription kit (Takara, Japan) was used to synthesize total RNA into cDNA. [21]The process of the reverse transcription reaction was as follows: 37°C for 15 minutes, 85°C for 5 seconds, and storage at 4˚C. To measure the expression of circular RNAs, linear RNA was degraded with RNase-R. Real-time PCR was performed with SYBR PreMix Ex Taq kit (Takara) and applied biological system (Foster City, CA). The thermal cycle conditions were set to 50˚C for 2 minutes, 95˚C for 10 minutes, followed by 40 cycles of 95˚C for 15 seconds, 60˚C for 10 minutes. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal control of circRNAs. [22]The fold change was calculated using the 2-ΔΔCT method. The primer sequences are listed in Table 1.
Table 1
Sequences of primers for Qrt-PCR
|
Name
|
|
Sequence
|
has_circRNA_101725
|
Forward
|
5'-TTCATCTCCTTCCTCAGC-3'
|
|
Reverse
|
5'-GCTGTTGTGGTGCCTGCT-3'
|
has_circRNA_000950
|
Forward
|
5'-ACCTCTATAAAACGGGAA-3'
|
|
Reverse
|
5'-AGGCAGAGCTGGGATTCA-3'
|
has_circRNA_405283
|
Forward
|
5'-TGTTACCAGCATGTTATC-3'
|
|
Reverse
|
5'-TTTTGCAGCCTTGTGTTA-3'
|
has_circRNA_048764
|
Forward
|
5'-TGCCCTCTCCCTGAAATA-3'
|
|
Reverse
|
5'-TCACGTTCTTGGTCACTT-3'
|
has_circRNA_406748
|
Forward
|
5'-TTTGTCCATCTCCCAAAC-3'
|
|
Reverse
|
5'-TTTCCGACATAACATCCT-3'
|
has_circRNA_005411
|
Forward
|
5'-GTCTCCCACATCACTCGG-3'
|
|
Reverse
|
5'-TGGAGTTGTAGAGCCAGG-3'
|
2.8 Statistical analyses
All statistical analyses were analyzed with SPSS 17.0(Chicago, IL, USA). The t-test was used for the comparison of samples between test and control groups and the Benjamini Hochberg FDR (the FDR cutoff was 0.05) was used for multiple-testing correction. P<0.05 was considered statistically significant.