Clinical samples
OLF tissues were harvested from three OLF patients and normal ligamentum flavum tissues from three non-OLF patients who received surgery for spine trauma was harvested as a control. This study was approved by the institutional review board of the Provincial Hospital Affiliated to Shandong University, and all patients signed informed consent. Diagnosis of OLF was performed by professor Da-chuan Wang based on clinical symptoms and radiological examinations (including X-ray, computed tomography and magnetic resonance imaging) of the spine.
Cell culture and treatments
Cells from the ligamentum flavum tissues were cultured as previously reported21.
Briefly, the ligament tissue was cut into small pieces under aseptic conditions and PBS
was used to wash the pieces three times to remove excess component. Next, 250 U/ml type 1A collagenase was used to digest the small pieces at 37℃and shake every five minutes. After the small pieces are completely digested,stop the digestion process using Dulbecco’s Modified Eagle’s medium (DMEM) and 10% fetal bovine serum (FBS). Finally, the cells were cultured in six wells with DMEM containing 10% FBS with 100 U/ml of penicillin and 100 µg/ml of streptomycin at cell culture incubator.
Osteogenic differentiation assay and Alizarin red staining
As we previous reported, we prepared osteogenic differentiation medium as follows: α-MEM with 10% fetal bovine serum, 10 mM β-glycerophosphate, 10nM dexamethasone, and 50 µg/mL ascorbic acid 22. Approximately 105 cells/well were were cultured in osteogenic differentiation medium. When the cells reached 60% confluence, the osteogenic differentiation medium was replaced with osteogenesis induction. After 21 days of induction, calci um mineral deposits were measured by Alizarin red staining using microscopy.
ALPstaining
ALP staining was used to evaluate the osteogenic effects as previously reported23. Approximately there were 105 cells/well in six well dishes. When the cells reached 60% confluence, the osteogenic differentiation medium was replaced with osteogenesis induction. After 7 days of induction, the induced cells were washed three times with PBS and then fixed with 4% formaldehyde for 20 minutes. An ALP staining solution was used to measure the ALP activity. After washing three times with PBS, the ALP activity was measured by microscopy.
Hematoxylin and Eosin (HE) staining assay
The HE staining was performed as we reported previously24. Briefly, the ossification ligament and normal ligament tissues were fixed in 4% paraformaldehyde at 4℃ for three days. Then the samples were embedded in paraffin and cut into 5-µm sections, deparaffinized in xylene, rehydrated through a series of graded ethanol, and washed in distilled water. Hematoxylin and Eosin (HE) staining was performed for histological observation.
Immunohistochemical (IHC) staining assay
In addition, IHC staining was used to evaluate the osteogenesis effect and the expression of IGF-2. The ossification ligament and normal ligament tissues were fixed in 4% paraformaldehyde at 4°C for three days. Then the samples were embedded in paraffin and cut into 5-µm sections,deparaffinized in xylene, rehydrated through a series of graded ethanol, and washed in distilled water. Subsequently, 3% hydrogen peroxide was used as a catalase quencher, and then the tissue sections were blocked in 10% goat serum (Sigma-Aldrich) for 1 hour. After washing with PBS for three times, an anti-IGF-2 antibody (1:100; Abcam; Cat. no. ab9574) was incubated with the sections at 4°C overnight, with non-immune serum as a control. Next, the tissue sections were incubated with a biotin-labeled secondary antibody (1:100; Proteintech; Cat. no. SA00004-6), and the signal was amplified and visualized with the chromogen diaminobenzidine, followed by hematoxylin counterstaining.
Immunofluorescent staining
The samples of OLF and normal ligamentum flavum tissues were obtained and cut into 5-µm sections. And then the sections were fixed at 4°C for 20 min. The sections were incubated with the anti-IGF-2 antibody (1:100; Abcam; Cat. no. ab9574) and anti-ALKBH5 (1:1000; Abcam; Cat. no. ab195377) antibody at 37°C for 2 hours. The fluorescently-labeled antibody was used to incubate with the sections at dark for 1 hour. After washed with three times, the sections were imaged using confocal microscope.
Vector construction and transfection
The human lentivirus-si-ALKBH5 (si-ALKBH5) and lentivirus-ALKBH5 (lv-ALKBH5) were purchased from Sangon Biotech (Shanghai, China). Puromycin was used to select the transfected cells for 7 days. The surviving cells were used as stable mass transfectants to perform the subsequent assays.
Dual Luciferase reporter assay
PCR product containing 300bp of the 3’-UTR of IGF-2 (wild-type and mutant) was first inserted into pGL3-basic vector. Then 200 ng pGL3-IGF-2(wild-type and mutant) promoter, along with 40 ng pRL-TK Vector (E2241, Promoga) were respectively transfected into the cells with over-expressed ALKBH5 or negative control using Lipofectamine 2000. The luciferase reporter assays were performed 48 hours after transfection using the Dual Luciferase Reporter Assay System (E1910, Promega).
Western blotting assay
Protein from the specimens were extracted using lysis buffer, and 20 ug of proteins was loaded into 10% SDS–PAGE gel and transferred to PVDF membranes (Millipore, Bedford, MA, USA). After blocking with 5% skimmed milk, the PVDF membranes were incubated with primary antibody including anti-ALKBH5 (1:1000; Abcam; Cat. no. ab195377), anti-IGF-2 antibody (1:1000; Abcam; Cat. no. ab9574) and GAPDH (1:1000; Abcam; Cat. no.ab9485) at 4℃ overnight. Next, the PVDF membranes were incubated with secondary antibody conjugated-horseradish peroxidase (1:1000; Proteintech; Cat. SA00001-2), and the bands were visualized using ECL detection reagents (Millipore).
Real-Time reverse transcriptase polymerase chain reaction
Real-time reverse transcriptase polymerase chain reaction (RT-PCR) was performed as we previously reported22. Trizol reagent (Invitrogen) was used to extract total RNA. PrimeScript RT reagent kit (TaKaRa, Japan) was used for reverse transcription. Real-time PCR analysis was performed using the SYBR Premix Ex Taq II kit (TaKaRa) and detected on the Roche LightCycler 480 sequence detection system. GAPDH were used as loading control for quantitation of mRNA.
Methylated RNA Immunoprecipitation and high-throughput RNA sequencing (MeRIP-seq)
MeRIP-Seq was performed by Cloudseq Biotech Inc. (Shanghai, China) according to the published procedure which was slight modifications. Briefly, the fragmented RNA was incubated with m6A polyclonal antibody (Synaptic Systems, 202003) in IPP buffer at 4°C for 2 hours. Then the mixture was immune-precipitated by incubation with protein-A beads (Thermo Fisher) at 4°C for an additional 2 hours. Then, bound RNA was eluted from the beads with N6-methyladenosine (BERRY & ASSOCIATES, PR3732) in IPP buffer and then extracted with Trizol reagent (Thermo Fisher) by following the manufacturer’s instruction. Purified RNA was used for RNA-seq library generation with NEBNext® Ultra™ RNA Library Prep Kit (NEB). Both the input sample without immunoprecipitation and the m6A IP samples were subjected to 150 bp paired-end sequencing on Illumina HiSeq sequencer. In addition, the q-PCR was performed to detect the level of m6A using purified RNA fragments.
Data of MeRIP-seq processing and bioinformatics analysis
Paired reads were harvested from Illumina HiSeq 4000 sequencer, and were quality controlled by Q30. After 3’ adaptor-trimming and low quality reads removing by cut adapt software (v1.9.3). First, clean reads of all libraries were aligned to the reference genome (UCSC HG19) by Hisat2 software (v2.0.4). ExomePeak software was used for identifying RNA m6A-modified regions (m6A peaks) with FDR (false discovery rate) < 0.05. As previously reported25, the common methylated RNAs (CMRs) were defined as RNAs containing at least one m6A peak in all samples, while the specific methylated RNAs (SMRs) were the RNAs with m6A modification only in one sample, and m6A peaks of SMRs were defined as specific m6A peaks. Biological process, molecular function and cellular component analysis were performed for the enrichment of differentially methylated genes, CMRs and SMRs.
Statistical analysis
All these experiments were repeated at least three times. The data were shown as means ± standard deviation (SD). Means of multiple groups were compared by one-way analysis of variance (with Fisher’s least significant difference [LSD] test). Statistical analysis was conducted in SPSS 20.0 software (IBM Corp., Armonk, NY, USA). Data with P values <0.05 was considered statistically significant.