Aberrant expression of microRNAs (miRNAs) has been associated with spinal ossification of the posterior longitudinal ligament (OPLL). Our initial bioinformatic analysis identified differentially expressed ADORA2A in OPLL and its regulatory miRNAs miR-497 and miR-195. Hence, this study was conducted to clarify the functional relevance of miR-497-195 cluster in OPLL, which may implicate in Adenosine A2A (ADORA2A). PLL tissues were collected from OPLL and non-OPLL patients, followed by quantification of miR-497, miR-195 and ADORA2A expression. The expression of miR-497, miR-195 and/or ADORA2A was altered in posterior longitudinal ligament (PLL) cells, which then were stimulated with cyclic mechanical stress (CMS). We validated that ADORA2A was expressed highly, while miR-497 and miR-195 were down-regulated in PLL tissues of OPLL patients. miR-195 and miR-497 expression in CMS-treated PLL cells was restored by a demethylation reagent 5-aza-2′-deoxycytidine (AZA). Moreover, expression of miR-195 and miR-497 was decreased by promoting promoter CpG island methylation. ADORA2A was verified as the target of miR-195 and miR-497. Overexpression of miR-195 and miR-497 diminished expression of osteogenic factors in PLL cells by inactivating the cAMP/PKA signaling pathway via down-regulation of ADORA2A. Collectively, miR-497-195 cluster augments osteogenic differentiation of PLL cells by inhibiting ADORA2A-dependent cAMP/PKA signaling pathway.

Introduction

Ossification of the posterior longitudinal ligament (OPLL) is a hyperostotic condition in the tissues of spinal ligament, which can result from non-genetic factors such as age, physical strain on the spinal ligament, diets and specific genetic factors [1]. OPLL is associated with many diseases including ankylosing spondylitis and some spondyloarthropathies. Moreover, OPLL can cause neurological deficits due to the compression of spinal cord, which can be treated by surgery [2]. It is interesting to note that the osteogenic differentiation of posterior longitudinal ligament (PLL)-derived fibroblasts is a significant factor underlying the pathogenesis of OPLL [3]. Moreover, a previous study has suggested that primary human ligament fibroblasts have been used to clarify the molecular mechanisms and identify potential therapeutic targets for OPLL treatment [4]. Therefore, to explore the molecular mechanisms underlying osteogenic differentiation in OPLL cells will help better understand the pathology of OPLL.

In this study, differential analysis was conducted in microarray data GSE5464 from GEO database, which observed that Adenosine A2A (ADORA2A) was differentially expressed in OPLL. In a prior research, ADORA2A receptor was reported to be associated with osteoblast differentiation [5], but the role of ADORA2A in OPLL remains to be defined.

Based on the analysis of TargetScan website, it was predicted that ADORA2A was a target gene of several microRNAs (miRNAs), including hsa-miR-497-5p and hsa-miR-195-5p. Many miRNAs have been proved to play pivotal roles in the onset and progression of OPLL [6]. For instance, the correlation of miR-10a with OPLL development has been elucidated in a prior study [7]. Previous literature substantiated that miR-497-195 cluster, a member of the miR-15 family, was associated with osteoblast differentiation [8].

Furthermore, ADORA2A receptor was proved to respond to luminal adenosine by regulating the cAMP/protein kinase (PKA) signaling pathway [9]. The relationship between ADORA2A and the cAMP/PKA signaling pathway was also explained in another research, which reported the activating ADORA2A could up-regulate cAMP and PKA levels [10].

Therefore, a hypothesis could be drawn that miR-497-195 cluster bound to ADORA2A to regulate the cAMP/PKA signaling pathway, hence affecting osteogenic differentiation of PLL cells. In the present study, we study OPLL by making research on the regulatory mechanism of miR-497-195 cluster, which is expected to act as a potent target for ameliorating OPLL.

Experimental procedures

Ethic statement

This study was ratified by the Ethics Committee of the 2nd Affiliated Hospital of Harbin Medical University. Each patient enrolled in this study provided the informed consent.

Sample collection

The patients with OPLL who were confirmed by computerized tomography and preoperative magnetic resonance imaging were enrolled, including 8 males, and 5 females, aged between 41 and 69 years old with a mean age of 50.85 ± 7.15 years old. The patients without OPLL (non-OPLL) who received cervical corpectomy due to spinal trauma were included, consisting of 7 males and 6 females (aged between 39 and 68 years old with a mean age of 52.08 ± 8.19 years old). From January 2017 to April 2018, 13 OPLL specimens were harvested from patients with OPLL during anterior cervical discectomy. Meanwhile, another 13 PLL specimens were obtained from non-OPLL patients during anterior cervical corpectomy as control.

Cell culture, identification and transfection

The clinically collected PLL tissues were rinsed two times with phosphate buffer saline to remove the attached ossification tissues avoiding any crush on the PLL tissues. In brief, specimens were minced into 0.3 cm3 pieces and cultured in the Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) in an incubator with 5% CO2 at 37°C. Passage was initiated when cell confluence reached ∼80%. The culture medium was regularly renewed and cells were detached with trypsin and passaged when they settled the bottom. The obtained cells were PLL fibroblasts, which were then under observation considering growth, morphology and purity. The PLL fibroblasts at passage 3 were selected for subsequent experiments.

The vimentin immunofluorescence staining was applied to identify fibroblasts [4]. The cultured cells were incubated with rabbit against vimentin antibody (ab193555, 1: 500, Abcam Inc., Cambridge, U.K.) overnight at 4°C, and then incubated with tetramethyl rhodamine isothiocyanate (TRITC)-labeled goat anti-rabbit Immunoglobulin G (IgG) antibody (ab6718, 1: 1000, Abcam) for 1 h at room temperature. The nucleus was stained with 4′,6-diamidino-2-phenylindole for 10 min.

ADORA2A recombinant overexpression plasmid was constructed by pcDNA3.1 and silenced ADORA2A recombinant plasmid was formed by pRNAT-U6.1/Neo, which were provided by GenePharma (Shanghai, China). Subsequently, miR-195/497 mimic and inhibitor were used for exogenous regulation of miR-195/497 expression. cAMP/PKA signaling pathway inhibitor H89 (10 µM, S1643) was purchased from Beyotime Biotechnology (Shanghai, China). Next, the transfection was conducted following the manufacturer's protocol of Lipofectamine 2000 (Invitrogen, Carlsbad, CA, U.S.A.). After 24-h transfection, cyclic mechanical stress (CMS) on cells was performed for 12 h every day by Flexercell-4000 tension system (Flexcell International Corp., Hillsborough, NC, U.S.A.) with 0.5-Hz frequency and 10% amplitude.

RNA isolation and quantitation

Total RNA was extracted using RNeasy Mini Kit (Qiagen, Valencia, CA, U.S.A.), and reversely transcribed into complementary DNA (cDNA) by reverse transcription (RT) kit (RR047A, TaKaRa, Tokyo, Japan). The RT of miRNA was conducted using the tailing method. The separated RNA was subjected to polyadenylation using NCode™ miRNA First-Strand cDNA Synthesis Kit (MIRC10, Invitrogen). Samples were loaded by SYBR Premix EX Taq kit (RR420A, TaKaRa) and assessed in a real-time fluorescence quantitative polymerase chain reaction (qPCR) (ABI7500, Applied Biosystems, Foster City, CA, U.S.A.). The primer for miRNA was the universal primer in the first strand cDNA synthesis kit, while other primers were synthesized by Sangon Biotech (Shanghai, China) (Table 1). The relative expression normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or U6 was calculated with the formula 2−ΔΔCt.

Table 1
Primer sequence for RT-qPCR
GenesPrimer sequences
miR-497 F: 5′-CAGCAGCACACTGTGGTTTGT-3′ 
miR-195 F: 5′-TAGCAGCACAGAAATATTGGC-3′ 
ADORA2A F: 5′-CGCTCCGGTACAATGGCTT-3′ 
R: 5′-TTGTTCCAACCTAGCATGGGA-3′ 
ALP F: 5′-CCAAGGACGCTGGGAAATCT-3′ 
R: 5′-TATGCATGAGCTGGTAGGCG-3′ 
Runx2 F: 5′-GCGCATTCCTCATCCCAGTA-3′ 
R: 5′-GGCTCAGGTAGGAGGGGTAA-3′ 
collagen I F: 5′-CAGGGCGACAGAGGCATAAAGG-3′ 
R: 5′-CCAACAGGACCAGCATCACCAG-3′ 
OCN F: 5′-ATGAGAGCCCTCACACTCCT-3′ 
R: 5′-CTTGGACACAAAGGCTGCAC-3′ 
GAPDH F: 5′-TCAAGGCTGAGAACGGGAAG-3′ 
R: 5′-TGGACTCCACGACGTACTCA-3′ 
U6 F: 5′-CTCGCTTCGGCAGCACA-3′ 
GenesPrimer sequences
miR-497 F: 5′-CAGCAGCACACTGTGGTTTGT-3′ 
miR-195 F: 5′-TAGCAGCACAGAAATATTGGC-3′ 
ADORA2A F: 5′-CGCTCCGGTACAATGGCTT-3′ 
R: 5′-TTGTTCCAACCTAGCATGGGA-3′ 
ALP F: 5′-CCAAGGACGCTGGGAAATCT-3′ 
R: 5′-TATGCATGAGCTGGTAGGCG-3′ 
Runx2 F: 5′-GCGCATTCCTCATCCCAGTA-3′ 
R: 5′-GGCTCAGGTAGGAGGGGTAA-3′ 
collagen I F: 5′-CAGGGCGACAGAGGCATAAAGG-3′ 
R: 5′-CCAACAGGACCAGCATCACCAG-3′ 
OCN F: 5′-ATGAGAGCCCTCACACTCCT-3′ 
R: 5′-CTTGGACACAAAGGCTGCAC-3′ 
GAPDH F: 5′-TCAAGGCTGAGAACGGGAAG-3′ 
R: 5′-TGGACTCCACGACGTACTCA-3′ 
U6 F: 5′-CTCGCTTCGGCAGCACA-3′ 

Note: miR-497, microRNA 497; miR-195, microRNA 195; ADORA2A, adenosine A2a receptor; ALP, aleurain-like protease; Runx2, runt related transcription factor 2; OCN, osteocalcin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; U6, U6 small nuclear RNA; RT-qPCR, reverse transcription quantitative polymerase chain reaction; F, forward; R, reverse.

Western blot analysis

Total protein was extracted from tissues or cells using Radio-Immunoprecipitation assay (RIPA) cell lysis buffer containing phenylmethanesulfonyl fluoride (PMSF). The proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride (PVDF) membrane. The membrane was incubated with diluted primary rabbit antibody (Abcam) to alkaline phosphatase (ALP) (ab83259, 1: 1000), Runx2 (ab23981, 1: 1000), collagen I (ab138492, 1: 1000), osteocalcin (OCN) (ab93876, 1: 500), phosphorylation of PKA (ab75991, 1: 2500), PKA (ab187515, 1: 1000), or GAPDH (ab181603, 1: 10 000) as loading control overnight at 4°C. The membrane was then incubated with horseradish peroxidase (HRP)-tagged secondary goat anti-rabbit IgG antibody (ab97051, 1: 2000, Abcam) for 1 h. Quantity One v4.6.2 software was employed to study the image obtained using Bio-Rad Image Analysis System (Bio-Rad Inc., Hercules, CA, U.S.A.). The relative protein expression was expressed as the ratio of protein band to be tested to the loading control band.

Dual-luciferase reporter gene assay

Wild type (WT) and Mutant type (Mut) reporter plasmids of ADORA2A (Wt-ADORA2A-3′untranslated region (UTR) and Mut-ADORA2A-3′UTR) were provided by GenePharma (Shanghai, China). Then, miR-497 mimic, miR-195 mimic or negative control (NC) of mimic were co-transfected with Wt-ADORA2A-3′UTR or Mut-ADORA2A-3′UTR into PLL cells. After 48 h, the following procedures were performed according to the instructions of luciferase detection kit (K801-200, BioVision, Milpitas, CA, U.S.A.). In the dual-luciferase reporter gene analysis system (Promega, Madison, WI, U.S.A.), the luciferase activity was detected. The light intensity from Renilla luciferase was normalized to that of firefly luciferase.

Methylation-specific PCR (MS-PCR)

Methylation of miR-195/497 promoter was determined by MS-PCR. First, genomic DNA was extracted according to the specification of Genomic DNA extraction kit (Beijing TIANGEN Biochemical Company, Beijing, China). DNA was stored at −80°C for further experiments after concentration and purity of genomic DNA were measured by ultraviolet spectrophotometry. Then, a total of 1 µg DNA was modified by bisulfite and stored at −80°C for no more than 1 month. Next, MS-PCR was performed following the detail as Herman et al. described [11]. Briefly, 20 µl PCR mixture contained 1 U MSP DNA Polymerase (2.5 U/µl), 1.6 µl dNTPs (2.5 mmol/L), 2 µl 10× MSP PCR Buffer, 2 µl primers (10 µmol/L) and 2 µl DNA template, which were increased to 20 µl by deionized water. MPS products were visualized by 2.5% electrophoretic agarose gels containing 0.55 mg/L ethidium bromide and analyzed by gel-imaging. Last, the density of methylation in miR-195/497 promoter region was assessed by checking the methylation status of CpG Island. Partial methylation was regarded as methylation level. The primers are depicted in Table 2.

Table 2
Methylation-specific PCR primer sequences
GenesPrimer sequences
miR-195/497-M F: 5′-AGAGATGGATTTTCGTTTAAAGTTC-3′ 
R: 5′-CCCAAACCAAATAAATCCTACG-3′ 
miR-195/497-U F: 5′-AGATGGATTTTTGTTTAAAGTTTGT-3′ 
R: 5′-CCAAACCAAATAAATCCTACACC-3′ 
GenesPrimer sequences
miR-195/497-M F: 5′-AGAGATGGATTTTCGTTTAAAGTTC-3′ 
R: 5′-CCCAAACCAAATAAATCCTACG-3′ 
miR-195/497-U F: 5′-AGATGGATTTTTGTTTAAAGTTTGT-3′ 
R: 5′-CCAAACCAAATAAATCCTACACC-3′ 

Notes: miR-195/497-M, methylated-specific primers for microRNA-195/497; miR-195/497-U, unmethylated-specific primers for microRNA-195/497; PCR, polymerase chain reaction; F, forward; R, reverse.

Chromatin immunoprecipitation (ChIP)

DNA-protein cross-link was disrupted into chromatin fragments (200–600 bp) by an ultrasonicator for 10 s each cycle (interval of 10 s; 15 cycles), followed with a centrifugation at 12 000×g and 4°C for 10 min. The supernatant was then incubated with NC IgG (5873S, 1: 20, Cell Signaling Technology, Boston, MA, U.S.A.), antibodies against DNA methyltransferase 1 DNMT1 (ab13537, 1: 50, Abcam) or DNMT3B (ab2851; 1: 50, Abcam) at 4°C overnight. DNA-protein complex was then precipitated by Pierce protein A/G Magnetic Beads (88803, Thermo Fisher Scientific Waltham, MA, U.S.A.). The DNA-protein complex was washed and de-cross-linked overnight at 65°C. DNA fragments were extracted by phenol/chloroform, and amplified for qPCR. Primer sequences were as follows: Forward: CCTTCCATTGTCCTGCGAC; Reverse: GGTGCAAAATAAACCCGCGC.

Enzyme linked immunosorbent assay (ELISA)

The PLL cells were lysed with 500 µl HCl (0.1 mol/L) at room temperature. Then cell lysate was collected, and the supernatant was harvested by 10-min centrifugation at 12 000 r/min at 4°C. cAMP concentration was detected under the instructions of cAMP Assay kit (ab234585, Abcam).

ALP staining and activity

ALP staining was conducted using NBT/BCIP staining kit (CoWin Biotech, Beijing, China). ALP activity was detected following the instructions of ALP activity colorimetric assay kit (BioVision). Briefly, PLL cells were lysed with 1% Triton X-100 (Sigma–Aldrich, St. Louis, MO, U.S.A.). The optical density (OD) value was detected at 405 nm. By using the Pierce protein assay kit (Thermo Fisher Scientific), total protein concentrations were determined by the bicinchoninic acid method. ALP activity was evaluated by the ratio of the absorbance levels relative to the protein concentration [12].

Alizarin red S (ARS) staining and quantification

Calcium nodule was confirmed by ARS staining. Then cells were stained with 0.1% ARS (pH = 4.2; Sigma–Aldrich) for 20 min. To quantify mineralized nodules, the staining resolution was dissolved in 1 ml 10% cetylpyridinium chloride (Sigma–Aldrich) for 1 h. OD value was detected at 570 nm by spectrophotometric method. The intensity of ARS was calculated by the ratio of the OD value to the total protein concentration [13].

Mechanical stimulation

The PLL cells were seeded into Flexercell plates (Flexcell Co., North Carolina, U.S.A.) at a density of 3 × 105 cells/well and cultured in DMEM containing 10% FBS for 24 h until cell confluence reached 70%, followed by further 24-h incubation in DMEM supplemented with 1% FBS. Subsequently, a Flexercell 4000 Strain Unit (Flexcell) was employed to generate cyclic tensile strain. With reference to the manufacturer's instructions, the cells were subjected to a cyclic tensile strain of 10% elongation at a frequency of 0.5 Hz for 12 and 24 h. The cells for control were cultured on the same plates without cyclic strain [4].

Statistical analysis

Statistical data were processed with SPSS 22.0 (SPSS, Inc., IBM, Armonk, NY, U.S.A.). Enumeration data were expressed as rate. Differences among groups were analyzed by Chi-square test or Fisher. Measurement data were described as mean ± standard deviation and with normal distribution and homogeneity of variance. Unpaired t-test was used for comparing two experimental groups, while differences among multiple groups were analyzed by one-way analysis of variance (ANOVA), followed by Tukey's post-hoc test. The correlation between miR-497/195 and ADORA2A in OPLL patients (n = 13) was determined by Pearson's correlation coefficient. A P < 0.05 indicated statistically significant.

Results

ADORA2A might be involved in OPLL

To identify differentially expressed genes (DEGs) in OPLL, microarray data GSE5464 of human spine PLL cells was obtained through Gene Expression Omnibus (GEO) database. After differential analysis of gene expression, 1897 genes with obviously differential expression were obtained (Supplementary Figure S1A), including 915 up-regulated genes and 982 down-regulated genes. Then Gene Ontology (GO) enrichment analysis was performed on DEGs, and enrichment terms with the minimum P value were chosen, which presented 96 differentially expressed genes located at regulation of anatomical structure size (GO: 0090066) (Supplementary Figure S1B). Protein-protein interaction (PPI) network of these genes was analyzed through STRING database, and visualized viaCytoscape software, which presented 52 genes with degree ≥ 5 (Supplementary Figure S1C). After comparing logFoldChange and p value of 52 genes, nine genes with the maximum degree of differential expression were screened out (Table 3). The expression of these 9 DEGs in OPLL (n = 13) and non-OPLL patients (n = 13) was evaluated using RT-qPCR (Figure 1A), showing that ADORA2A was expressed at a high level with the highest differential degree. Then, the differential analysis of ADORA2A was performed on the microarray dataset GSE5464, which displayed that ADORA2A expression was much higher in the OPLL patients than in the normal patients (P < 0.05; Figure 1B). To verify our bioinformatics analysis, ADORA2A expression was detected in 13 cases of PLL tissues from OPLL patients and 13 cases of PLL tissues from non-OPLL patients (control) using RT-qPCR (Figure 1C) and Western blot analysis (Figure 1D, randomly-selected five representative samples). It was revealed that in comparison with PLL tissues from non-OPLL patients, ADORA2A expression in PLL tissues of OPLL patients was obviously enhanced at both mRNA and protein levels (P < 0.05). Taken together, high expression of ADORA2A was found in PLL tissues of OPLL patients, indicating that ADORA2A may be involved in the OPLL development.

ADORA2A is highly expressed in PLL tissues from patients with OPLL.

Figure 1.
ADORA2A is highly expressed in PLL tissues from patients with OPLL.

(A) Expression of 9 DEGs in the PLL tissues of OPLL (n = 13) and non-OPLL (n = 13) patients determined using RT-qPCR. (B) Differential expression of ADORA2A in OPLL and non-OPLL patients predicted in microarray GSE5464. (C) ADORA2A mRNA expression in PLL tissues from non-OPLL (n = 13) and OPLL (n = 13) patients measured by RT-qPCR. (D) ADORA2A protein expression in PLL tissues from non-OPLL (n = 13) and OPLL patients (n = 13) normalized to GAPDH assessed by Western blot analysis (five most representative images are shown). * P < 0.05 vs. PLL tissues from non-OPLL patients. The measurement data were expressed as mean ± standard deviation. Unpaired t-test was used for comparison between two groups.

Figure 1.
ADORA2A is highly expressed in PLL tissues from patients with OPLL.

(A) Expression of 9 DEGs in the PLL tissues of OPLL (n = 13) and non-OPLL (n = 13) patients determined using RT-qPCR. (B) Differential expression of ADORA2A in OPLL and non-OPLL patients predicted in microarray GSE5464. (C) ADORA2A mRNA expression in PLL tissues from non-OPLL (n = 13) and OPLL (n = 13) patients measured by RT-qPCR. (D) ADORA2A protein expression in PLL tissues from non-OPLL (n = 13) and OPLL patients (n = 13) normalized to GAPDH assessed by Western blot analysis (five most representative images are shown). * P < 0.05 vs. PLL tissues from non-OPLL patients. The measurement data were expressed as mean ± standard deviation. Unpaired t-test was used for comparison between two groups.

Table 3
Genes with differential expression
GeneslogFCAveExprTP-Value
GMFG 6.117806 2.516316 4.975746 0.005863 
NPPA 5.373563 1.406894 2.963503 0.036581 
ADCY6 4.634161 −0.39231 3.388527 0.023627 
UTS2 4.179369 0.155153 5.209126 0.00491 
PPP1CA 4.169078 0.837992 8.466253 0.000681 
PLEK 4.035913 0.542194 5.896826 0.00301 
WAS 3.798859 0.809408 6.425514 0.00213 
ADORA2A 3.525588 1.172425 6.991764 0.001509 
NCKAP1 3.493725 1.294763 3.387256 0.023657 
GeneslogFCAveExprTP-Value
GMFG 6.117806 2.516316 4.975746 0.005863 
NPPA 5.373563 1.406894 2.963503 0.036581 
ADCY6 4.634161 −0.39231 3.388527 0.023627 
UTS2 4.179369 0.155153 5.209126 0.00491 
PPP1CA 4.169078 0.837992 8.466253 0.000681 
PLEK 4.035913 0.542194 5.896826 0.00301 
WAS 3.798859 0.809408 6.425514 0.00213 
ADORA2A 3.525588 1.172425 6.991764 0.001509 
NCKAP1 3.493725 1.294763 3.387256 0.023657 

Notes: GMFG, glia maturation factor gamma; NPPA, natriuretic peptide A; ADCY6, adenylate cyclase 6; UTS2, urotensin 2; PPP1CA, protein phosphatase 1 catalytic subunit alpha; PLEK, pleckstrin; WAS, WASP actin nucleation promoting factor; ADORA2A, adenosine A2a receptor; NCKAP1, NCK associated protein 1; FC, fold change; AveExpr, average expression.

ADORA2A knockdown suppresses osteogenic differentiation of PLL cells of OPLL patients

With an attempt to explore effects of ADORA2A on osteogenic differentiation of PLL cells, PLL cells were isolated from OPLL patients. The morphology of the isolated cells was mainly fusiform and polygonal star (Figure 2A). Immunofluorescence staining of vimentin was performed to identify the isolated cells, which showed that the expression of cytoplasmic vimentin was positive after TRITC labeling in isolated cells (Figure 2B). Thus, PLL fibroblasts were successfully isolated.

Silencing ADORA2A inhibits osteogenic differentiation of PLL cells.

Figure 2.
Silencing ADORA2A inhibits osteogenic differentiation of PLL cells.

(A) Images of PLL cells from OPLL patients after in vitro culture (×400). (B) PLL fibroblasts identified by vimentin immunofluorescence staining (×400). (C) Silencing efficiency of ADORA2A determined by RT-qPCR and Western blot analysis. (D) ALP activity in PLL cells analyzed by ALP staining (×200). (E) Calcium content in PLL cells measured by ARS staining (×200) and spectrophotometry. (F) Expression of osteogenic factors (ALP, Runx2, collagen I and OCN) in PLL cells determined by RT-qPCR and Western blot analysis. All the statistical data were measurement data and expressed as mean ± standard deviation. Comparisons among multiple groups (panel C) were performed using one-way ANOVA, followed by Tukey's post-hoc test. Differences between two groups (panels DF) were analyzed by unpaired t-test. * P < 0.05 vs. treatment with si-NC. The experiment was conducted at least three times independently.

Figure 2.
Silencing ADORA2A inhibits osteogenic differentiation of PLL cells.

(A) Images of PLL cells from OPLL patients after in vitro culture (×400). (B) PLL fibroblasts identified by vimentin immunofluorescence staining (×400). (C) Silencing efficiency of ADORA2A determined by RT-qPCR and Western blot analysis. (D) ALP activity in PLL cells analyzed by ALP staining (×200). (E) Calcium content in PLL cells measured by ARS staining (×200) and spectrophotometry. (F) Expression of osteogenic factors (ALP, Runx2, collagen I and OCN) in PLL cells determined by RT-qPCR and Western blot analysis. All the statistical data were measurement data and expressed as mean ± standard deviation. Comparisons among multiple groups (panel C) were performed using one-way ANOVA, followed by Tukey's post-hoc test. Differences between two groups (panels DF) were analyzed by unpaired t-test. * P < 0.05 vs. treatment with si-NC. The experiment was conducted at least three times independently.

Then, 3 independent siRNAs were designed to silence ADORA2A expression in PLL cells (Figure 2C). According to RT-qPCR and Western blot analysis, si-ADORA2A-2 exhibited the highest silence efficiency. Therefore, si-ADORA2A-2 was selected for the following study. Then, ADORA2A was silenced in PLL cells, which were then stimulated with 10% 0.5 Hz CMS (10% amplitude) for 12 h every day, and non-treatment with CMS was set as controls. After 7-day stimulation, ALP activity was evaluated (Figure 2D), which revealed that ALP activity in controls showed no significant difference (P > 0.05), while ALP activity was decreased in CMS-stimulated cells treated with si-ADORA2A. After 14-day stimulation, calcium nodule was stained with ARS, and calcium content was quantified by spectrophotometry (Figure 2E). It was displayed that ARS staining degree in controls showed no obvious difference (P > 0.05), while the color of ARS became lighter and calcium content was significantly decreased in CMS-stimulated cells treated with si-ADORA2A compared with CMS-stimulated cells treated with si-NC (P < 0.05). Meanwhile, expression of osteogenic factors (ALP, Runx2, collagen I and OCN) was determined by RT-qPCR and Western blot analysis. As described in Figure 2F, expression of ALP, Runx2, collagen I and OCN was overtly decreased by treatment of si-ADORA2A. In conclusion, knockdown of ADORA2A expression could repress osteogenic differentiation of PLL cells.

miR-195-497 cluster targets and down-regulates ADORA2A

To unravel how the differential expression of ADORA2A was regulated in OPLL, we intersected the genes targeted by miR-497 and miR-195 retrieved from the miDIP, miRDB, TargetScan and microT databases with the highly expressed DEGs obtained from GSE5464 microarray-based analysis. Besides, TargetScan website was utilized to predict the possible binding site between miR-195/497 and ADORA2A (Supplementary Figure S2A,B). Then miR-497 and miR-195 expression were detected in PLL tissues from patients with OPLL of the cervical spine and non-OPLL patients by RT-qPCR (Figure 3A), which showed that the levels of miR-497 and miR-195 in PLL tissues were lower in patients with OPLL of the cervical spine than in non-OPLL patients (P < 0.05). Then, according to Pearson's correlation analysis, in PLL tissues from 13 patients with OPLL of the cervical spine, the expression of miR-497 and miR-195 were negatively correlated with ADORA2A expression, and the expression of miR-497 was positively correlated with that of miR-195 (Figure 3B). Next, miR-497 and miR-195 expression in CMS-stimulated PLL cells from non-OPLL patients was determined by RT-qPCR (Figure 3C), displaying that CMS stimulation diminished miR-497 and miR-195 expression in PLL cells.

ADORA2A is a target gene of both miR-497 and miR-195.

Figure 3.
ADORA2A is a target gene of both miR-497 and miR-195.

(A) miR-497 and miR-195 expression in PLL tissues from OPLL and non-OPLL patients determined by RT-qPCR. * P < 0.05 vs. PLL tissues in non-OPLL patients; n = 13. (B) Correlation of the expression of miR-195 and miR-497 with ADORA2A in PLL tissues from patients with OPLL of the cervical spine (n = 13) analyzed using Pearson's correlation coefficient. (C) miR-195 and miR-497 expression in PLL cells after CMS treatments. * P < 0.05 vs. cell without CMS. (D) Targeting relationship of miR-497 and miR-195 with ADORA2A detected by dual-luciferase reporter gene assay. * P < 0.05 vs. mimic NC. (E) ADORA2A expression in PLL cells after treatment with miR-497 mimic and miR-195 mimic. * P < 0.05 vs. mimic NC. The measurement data were described as mean ± standard deviation. In panel B, unpaired t-test was conducted to compare the difference between two experimental groups. Comparisons among multiple groups (panels C and D) were analyzed by one-way ANOVA, followed by Tukey's post-hoc test. The experiment was repeated three times independently.

Figure 3.
ADORA2A is a target gene of both miR-497 and miR-195.

(A) miR-497 and miR-195 expression in PLL tissues from OPLL and non-OPLL patients determined by RT-qPCR. * P < 0.05 vs. PLL tissues in non-OPLL patients; n = 13. (B) Correlation of the expression of miR-195 and miR-497 with ADORA2A in PLL tissues from patients with OPLL of the cervical spine (n = 13) analyzed using Pearson's correlation coefficient. (C) miR-195 and miR-497 expression in PLL cells after CMS treatments. * P < 0.05 vs. cell without CMS. (D) Targeting relationship of miR-497 and miR-195 with ADORA2A detected by dual-luciferase reporter gene assay. * P < 0.05 vs. mimic NC. (E) ADORA2A expression in PLL cells after treatment with miR-497 mimic and miR-195 mimic. * P < 0.05 vs. mimic NC. The measurement data were described as mean ± standard deviation. In panel B, unpaired t-test was conducted to compare the difference between two experimental groups. Comparisons among multiple groups (panels C and D) were analyzed by one-way ANOVA, followed by Tukey's post-hoc test. The experiment was repeated three times independently.

Subsequently, targeting relationship between miR-497 and ADORA2A, as well as between miR-195 and ADORA2A were detected by dual-luciferase reporter assay in PLL cells (Figure 3D). It was suggested that the co-transfection of miR-195 mimic or miR-497 mimic decreased the luciferase activity of WT-ADORA2A-3′UTR, while the luciferase activity of Mut-ADORA2A-3′UTR was not affected (P > 0.05). Furthermore, RT-qPCR and Western blot analysis exhibited that ADORA2A expression was lowered in PLL cells treated with miR-497 mimic or miR-195 mimic (Figure 3E). We could conclude from the findings above that miR-497 and miR-195 could target and down-regulate ADORA2A.

Promoter methylation of miR-195 and miR-497 negatively regulates their expression

Since the expression of miR-195 and miR-497 were lower in patients with OPLL, we further decided to explore how their expression was regulated. After CpG island was discovered in miR-195/497 promoter region using MethPrimer website (Supplementary Figure S3A), we aimed to evaluate the correlation of miR-195 and miR-497 expression with their promoter methylation. MSP was employed to test the methylation level of CpG island in the promoter region of miR-195 and miR-497 in PLL tissues from non-OPLL patients and patients with OPLL of the cervical spine (Supplementary Figure S3B). In patients with OPLL of the cervical spine, CpG island methylation in miR-195 and miR-497 promoter was increased in PLL tissues in contrast with that in non-OPLL patients (P < 0.05). Furthermore, MSP was performed to examine CpG island methylation level in the promoters of miR-195 and miR-497 in CMS-stimulated non-OPLL and OPLL cells. As depicted in Figure 4A, CMS treatment obviously enhanced the CpG island methylation in the promoters of miR-195 and miR-497 in PLL cells. Then, PLL cells were stimulated with CMS and treated with the DNA-hypomethylating agent 5-aza-2′-deoxycytidine (AZA) or dimethylsulfoxide (DMSO) respectively. MSP demonstrated that the CpG island methylation in DMSO-treated control cells showed no significant change (P > 0.05), while CpG island methylation in CMS-stimulated cells was distinctly diminished after treatment with AZA (Figure 4B). Next, miR-497, miR-195 and ADORA2A expression in PLL cells treated with AZA were examined using RT-qPCR (Figure 4C). Treatment of AZA significantly increased the expression of miR-497 and miR-195 but diminished the expression of ADORA2A in CMS-treated PLL cells. Enrichments of DNMT1 and DNMT3B in miR-195 and miR-497 promoter of CMS-treated PLL cells were measured by ChIP assay (Figure 4D). The result exhibited that DNMT1 and DNMT3B enrichments in control PLL cells showed no significant difference (P > 0.05). However, DNMT1 and DNMT3B enrichments were distinctly enhanced in PLL cells by CMS treatment. Therefore, the expression of miR-195 and miR-497 was negatively correlated with the CpG island methylation level in the promoter. CMS treatment decreased the expression of miR-195 and miR-497 in PLL cells through up-regulating promoter methylation.

The expression of miR-195 and miR-497 is negatively correlated with the CpG island methylation in their promoter.

Figure 4.
The expression of miR-195 and miR-497 is negatively correlated with the CpG island methylation in their promoter.

(A) CpG island methylation in OPLL and non-OPLL cells treated with CMS. (B) CpG island methylation in PLL cells treated with AZA; C, miR-497, miR-195 and ADORA2A expression in CMS-treated PLL cells after treatment of AZA examined by RT-qPCR. * P < 0.05 vs. CMS-treated PLL cells with DMSO. (D) The enrichment of DNMT1 and DNMT3B in miR-195 and miR-497 promoter of PLL cells treated with CMS measured by ChIP assay. * P < 0.05 vs. IgG. The measurement data were described as mean ± standard deviation. Unpaired t-test was used for analyzing differences between two groups (panel C); one-way ANOVA was employed to analyze difference in multiple groups (panel D), with Tukey's post-hoc test. The experiment was carried out at least three times independently. U, unmethylated; M, Methylated.

Figure 4.
The expression of miR-195 and miR-497 is negatively correlated with the CpG island methylation in their promoter.

(A) CpG island methylation in OPLL and non-OPLL cells treated with CMS. (B) CpG island methylation in PLL cells treated with AZA; C, miR-497, miR-195 and ADORA2A expression in CMS-treated PLL cells after treatment of AZA examined by RT-qPCR. * P < 0.05 vs. CMS-treated PLL cells with DMSO. (D) The enrichment of DNMT1 and DNMT3B in miR-195 and miR-497 promoter of PLL cells treated with CMS measured by ChIP assay. * P < 0.05 vs. IgG. The measurement data were described as mean ± standard deviation. Unpaired t-test was used for analyzing differences between two groups (panel C); one-way ANOVA was employed to analyze difference in multiple groups (panel D), with Tukey's post-hoc test. The experiment was carried out at least three times independently. U, unmethylated; M, Methylated.

miR-195 and miR-497 inhibits the cAMP/PKA signaling pathway to suppress osteogenic differentiation of PLL cells by targeting ADORA2A

Since ADORA2A has been reported to be able to up-regulate cAMP and PKA levels, we further intended to explore whether ADORA2A affected osteogenic differentiation through activating the cAMP/PKA signaling pathway. First, we examined whether ADORA2A could regulate the cAMP/PKA signaling pathway in PLL cells. ADORA2A, miR-195 or miR-497 was overexpressed in PLL cells, which were then stimulated with CMS 7 days after stimulation. cAMP concentration in PLL cells was detected using ELISA (Figure 5A). It was demonstrated that miR-195 mimic or miR-497 mimic enhanced cAMP level, which could be blocked by oe-ADORA2A (P < 0.05). Then, Western blot analysis was performed to assess expression of osteogenic factors (ALP, Runx2, collagen I and OCN) and phosphorylation of PKA in PLL cells (Figure 5B). Expression of ALP, Runx2, collagen I and OCN and phosphorylation of PKA were increased in PLL cells treated with miR-195 mimic /miR-497 mimic, which could be prevented by oe-ADORA2A (P < 0.05). Next, ALP activity was analyzed (Figure 5C), which illustrated that ALP staining and activity were significantly elevated when cells were treated with miR-195 mimic/miR-497 mimic, which could be reversed by oe-ADORA2A (P < 0.05). After 14-day CMS stimulation, calcium nodule was stained using ARS and the calcium content was quantified by spectrophotometry (Figure 5D). Furthermore, ARS staining was increased and the content of calcium was obviously enhanced in response to miR-195 mimic/miR-497 mimic, which could be blocked by oe-ADORA2A (P < 0.05).

miR-195 and miR-497 suppress osteogenic differentiation of PLL cells via inactivation of the cAMP/PKA signaling pathway by targeting ADORA2A.

Figure 5.
miR-195 and miR-497 suppress osteogenic differentiation of PLL cells via inactivation of the cAMP/PKA signaling pathway by targeting ADORA2A.

(A) cAMP concentration in PLL cells determined by ELISA. (B) Expression of osteogenic factors and phosphorylation of PKA in PLL cells. (C) ALP staining (×200) and activity in PLL cells. (D) Calcium nodule stained by ARS (×200) and calcium content in PLL cells quantified by spectrophotometry. (E) PLL cells were treated with inhibitor NC, miR-497 inhibitor or miR-195 inhibitor, or treated with miR-497 inhibitor +H89 or miR-195 inhibitor +H89. The first three groups were also treated with DMSO. After 24 h, CMS stimulation was performed. Phosphorylation of PKA and ALP, Runx2, collagen I and OCN expression in PLL cells were measured. (F) ALP staining (×200) and activity in PLL cells. (G) Calcium nodule stained by ARS (×200) and calcium content in PLL cells quantified by spectrophotometry. The data were measurement data and described as mean ± standard deviation. Differences among multiple groups were analyzed by one-way ANOVA, followed by Tukey's post-hoc test. In panels AD, * P < 0.05 vs. mimic NC + oe-NC; # P < 0.05 vs. mimic NC + oe-ADORA2A, while in panels EG, * P < 0.05 vs. miR-497 inhibitor; # P < 0.05 vs. miR-195 inhibitor. The experiment was carried out thrice independently.

Figure 5.
miR-195 and miR-497 suppress osteogenic differentiation of PLL cells via inactivation of the cAMP/PKA signaling pathway by targeting ADORA2A.

(A) cAMP concentration in PLL cells determined by ELISA. (B) Expression of osteogenic factors and phosphorylation of PKA in PLL cells. (C) ALP staining (×200) and activity in PLL cells. (D) Calcium nodule stained by ARS (×200) and calcium content in PLL cells quantified by spectrophotometry. (E) PLL cells were treated with inhibitor NC, miR-497 inhibitor or miR-195 inhibitor, or treated with miR-497 inhibitor +H89 or miR-195 inhibitor +H89. The first three groups were also treated with DMSO. After 24 h, CMS stimulation was performed. Phosphorylation of PKA and ALP, Runx2, collagen I and OCN expression in PLL cells were measured. (F) ALP staining (×200) and activity in PLL cells. (G) Calcium nodule stained by ARS (×200) and calcium content in PLL cells quantified by spectrophotometry. The data were measurement data and described as mean ± standard deviation. Differences among multiple groups were analyzed by one-way ANOVA, followed by Tukey's post-hoc test. In panels AD, * P < 0.05 vs. mimic NC + oe-NC; # P < 0.05 vs. mimic NC + oe-ADORA2A, while in panels EG, * P < 0.05 vs. miR-497 inhibitor; # P < 0.05 vs. miR-195 inhibitor. The experiment was carried out thrice independently.

Thereafter, to probe whether inhibition of the cAMP/PKA signaling pathway would affect the effect of miR-195/497-ADORA2A axis on osteogenic differentiation of PLL cells, the inhibitor of cAMP/PKA signaling pathway H89 was employed to inactivate the cAMP/PKA signaling pathway. Then, PLL cells were treated with inhibitor NC, miR-497 inhibitor or miR-195 inhibitor, or treated with miR-497 inhibitor +H89 or miR-195 inhibitor +H89. The first three groups were also treated with DMSO. After 24 h, CMS stimulation was performed. After 7-day stimulation, phosphorylation of PKA and expression of ALP, Runx2, collagen I and OCN were determined using Western blot analysis (Figure 5E). Phosphorylation of PKA and expression of ALP, Runx2, collagen I and OCN were overtly reduced after the treatment of miR-497/miR-195 inhibitor +H89 in comparison with the treatment with miR-497/miR-195 inhibitor (P < 0.05). Next, ALP staining was carried out (Figure 5F), which indicated that compared with cells treated with miR-497/miR-195 inhibitor, ALP staining and activity were markedly reduced in cells treated with miR-497/miR-195 inhibitor +H89 (P < 0.05). After 14-day stimulation, calcium nodule was then stained using ARS and calcium content was quantified by spectrophotometry (Figure 5G). It was demonstrated that ARS staining and calcium content were diminished in cells treated with miR-497/miR-195 inhibitor +H89 in contrast with cells treated with miR-497/miR-195 inhibitor (P < 0.05). Collectively, miR-195 and miR-497 represses the cAMP/PKA signaling pathway and then decreases osteogenic differentiation of PLL cells by down-regulating ADORA2A.

Discussion

Previous work defined OPLL of the cervical spine as a spinal dysfunction which could give rise to more serious problems such as radiculopathy and myelopathy [13]. Therefore, early diagnosis of OPLL is necessary. However, current diagnosis of OPLL is frequently linked to computerized tomography which may result in radiation-induced impairment, while miRNAs are regarded as a less nociceptive diagnostic marker which cause no impairment [14]. Therefore, we sought to investigate more deeply the role of miRNAs in OPLL for the sake of understanding pathology, diagnosis and prognosis of OPLL of the cervical spine. Our data unraveled that methylation-mediated down-regulation of miR-195 and miR-497 promoted osteogenic differentiation of PLL cells by activating the cAMP/PKA signaling pathway via ADORA2A.

We reasoned that miR-497 and miR-195 were down-regulated in PLL tissues of OPLL patients. As explained previously, miR-497 was investigated in the process of bone formation, namely endochondral ossification, in which miR-497 expression was distinctly diminished [15]. It was also reported that miR-195-5p was expressed poorly during osteogenic differentiation of PLL cells; at a deeper level, overexpressing miR-195-5p inhibited osteogenic differentiation in the context of mechanical loading [16]. Meanwhile, miR-497-195 cluster was demonstrated to be a prospective target in osteoporosis therapy [17]. However, it was reported that miR-497-195 cluster was up-regulated in differentiation of calvarial osteoblasts, which contradicted our finding [18]. The difference may result from the nature of miR-497-195 cluster expression, which was dependent on several factors including differentiation stage, cell type or microenvironment [8].

Moreover, we also found that the low expression of miR-195 and miR-497 was attributed to the presence of promoter CpG island methylation, and that miR-195 and miR-497 expression in CMS-treated PLL cells was restored by the DNMT inhibitor AZA. It was documented that methylation of CpG islands generally occurred in silenced genes and hypomethylation was observed in the regulatory areas of transcriptionally active genes. Furthermore, miRNA expression can be down-regulated by DNA methylation and histone modifications [19]. As a DNMT inhibitor, AZA was often utilized to explore DNA methylation [20]. Inverse correlation between miRNA expression and DNA methylation was previously elucidated. For instance, a prior study revealed that miR-195 and miR-378 expression was impaired in gastric cancer cells by DNA methylation in their promoters [21]. Additionally, prior researches also demonstrated that miR-195 and miR-497 were poorly expressed in precancerous colorectal lesions due to hypermethylation [22,23], which supported our results.

Our current finding that ADORA2A expressed highly in OPLL of the cervical spine was expanded in a prior work which stated that activating ADORA2A stimulated bone formation [24]. Consistently, a research conducted by BorzoGharibi et al. [5] elucidated that ADORA2A was up-regulated during osteogenic differentiation of MSCs, which indicated that ADORA2A was critical to osteogenic differentiation. Concerning ADORA2A clinical value, previous work reported that inhibiting ADORA2A could be a more effective and safer way to ameliorate spinal injury [25]. In our study, ADORA2A knockdown was proved to significantly attenuate osteogenic differentiation of spinal PLL cells, which provided an in-depth thought on application of ADORA2A in therapy.

In this study, we employed cAMP/PKA signaling pathway inhibitor H89 to observe the effect of the cAMP/PKA signaling pathway on OPLL and unraveled that inactivation of the cAMP/PKA signaling pathway was able to decrease osteogenic differentiation of PLL cells, which was in general agreement with prior work that activation of the cAMP/PKA signaling pathway induced differentiation of osteoblastic cells [26]. Specifically, we found that cAMP/PKA was regulated by ADORA2A. Inhibition of ADORA2A led to cAMP/PKA signaling pathway suppression, therefore lessening osteogenic differentiation of spinal PLL cells. Consistent with our study, ADORA2B was proved to activate the cAMP/PKA signaling pathway and give rise to highly-expressed osteogenic genes including Runx2 and OCN [27]. Also, another study illustrated the promoting effects of ADORA2A on the cAMP/PKA signaling pathway [28].

Thus far, this study argued that methylation of miR-497-195 cluster relieved the inhibition on ADORA2A to activate the cAMP/PKA signaling pathway, which finally induced osteogenic differentiation of PLL cells (Supplementary Figure S4). Our study provided more clues in understanding the mechanisms underlying bone formation, spinal injuries or other osteogenic differentiation-related diseases.

Competing Interests

The authors declare that there are no competing interests associated with the manuscript.

Funding

This study was supported by the Fund of the 2nd Affiliated Hospital of Harbin Medical University [KYCX2019-03] and Postdoctoral Startup Fund of Heilongjiang Province [LBH-Q19034] and Guangdong Basic and Applied Basic Research Foundation [2020A1515010171].

Author Contribution

A.J., N.W., Y.J. and X.Y. designed the study. G.C. collated the data, designed and developed the database, carried out data analyses and produced the initial draft of the manuscript. H.C., P.K., H.R. and Y.J. contributed to drafting the manuscript. S.X. and J.Y. checked and edited the manuscript and contributed to the revision. All authors have read and approved the final submitted manuscript.

Acknowledgements

The authors sincerely appreciate all members participated in this work.

Abbreviations

     
  • ALP

    alkaline phosphatase

  •  
  • ARS

    Alizarin red S

  •  
  • CMS

    cyclic mechanical stress

  •  
  • DMEM

    Dulbecco's Modified Eagle Medium

  •  
  • FBS

    fetal bovine serum

  •  
  • MSCs

    mesenchymal stem cells

  •  
  • OPLL

    ossification of the posterior longitudinal ligament

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Author notes

*

These authors are regarded as co-first authors.

Supplementary data