Dynamic chromatin accessibility tuning by the long noncoding RNA ELDR accelerates chondrocyte senescence and osteoarthritis.

Epigenetic reprogramming plays a critical role in chondrocyte senescence during osteoarthritis (OA) pathology, but the underlying molecular mechanisms remain to be elucidated. Here, using large-scale individual datasets and genetically engineered (Col2a1-CreERT2;Eldrflox/flox and Col2a1-CreERT2;ROSA26-LSL-Eldr+/+ knockin) mouse models, we show that a novel transcript of long noncoding RNA ELDR is essential for the development of chondrocyte senescence. ELDR is highly expressed in chondrocytes and cartilage tissues of OA. Mechanistically, exon 4 of ELDR physically mediates a complex consisting of hnRNPL and KAT6A to regulate histone modifications of the promoter region of IHH, thereby activating hedgehog signaling and promoting chondrocyte senescence. Therapeutically, GapmeR-mediated silencing of ELDR in the OA model substantially attenuates chondrocyte senescence and cartilage degradation. Clinically, ELDR knockdown in cartilage explants from OA-affected individuals decreased the expression of senescence markers and catabolic mediators. Taken together, these findings uncover an lncRNA-dependent epigenetic driver in chondrocyte senescence, highlighting that ELDR could be a promising therapeutic avenue for OA.

Precise targeting of miR-141/200c cluster in chondrocytes attenuates osteoarthritis development.

OBJECTIVES: Despite preclinical studies involving miRNA therapeutics conducted in osteoarthritis (OA) over the years, none of these miRNAs have yet translated to clinical applications, owing largely to the lack of efficient intra-articular (IA) delivery systems. Here, we investigated therapeutic efficacy of the chondrocyte-specific aptamer-decorated PEGylated polyamidoamine nanoparticles (NPs)-based miRNAs delivery for OA. METHODS: The role of miR-141/200c cluster during skeletal and OA development was examined by miR-141/200cflox/flox mice and Col2a1-CreERT2; miR-141/200cflox/flox mice. Histological analysis was performed in mouse joints and human cartilage specimens. Chondrocyte-specific aptamer-decorated NPs was designed, and its penetration, stability and safety were evaluated. OA progression was assessed by micro-CT analysis, X-ray and Osteoarthritis Research Society International scores after destabilising the medial meniscus surgery with miR-141/200c manipulation by NPs IA injection. Mass spectrometry analysis, molecular docking and molecular dynamics simulations were performed to investigate the interaction between aptamer and receptor. RESULTS: Increased retention of NPs inside joint space is observed. The NPs are freely and deeply penetrant to mice and human cartilage, and unexpectedly persist in chondrocytes for at least 5 weeks. OA chondrocytes microenviroment improves endo/lysosomal escape of microRNAs (miRNAs). Therapeutically, IA injection of miR-141/200c inhibitors provides strong chondroprotection, whereas ectopic expression of miR-141/200c exacerbates OA. Mechanistically, miR-141/200c promotes OA by targeting SIRT1, which acetylates histone in the promoters of interleukin 6 (IL-6), thereby activating IL-6/STAT3 pathway. CONCLUSIONS: Our findings indicate that this nanocarrier can optimise the transport kinetics of miR-141/200c into chondrocytes, fostering miRNA-specific disease-modifying OA drugs development.

MicroRNA-218-5p as a Potential Target for the Treatment of Human Osteoarthritis.

Emerging evidence suggests that dysregulated microRNAs (miRNAs) play a pivotal role in osteoarthritis (OA), but the role of specific miRNAs remains unclear. Accordingly, we identified OA-associated miRNAs and functional validation of results. Here, we demonstrate that miR-218-5p is significantly upregulated in moderate and severe OA and correlates with scores on a modified Mankin scale. Through gain-of-function and loss-of-function studies, miR-218-5p was shown to significantly affect matrix synthesis gene expression and chondrocyte proliferation and apoptosis. Using SW1353 and C28/I2 cells, PIK3C2A mRNA was identified as a target of miR-218-5p. Downregulation of miR-218-5p dramatically promoted expression of PIK3C2A and its downstream target proteins, such as Akt, mTOR, S6, and 4EBP1. More importantly, OA mice exposed to a miR-218-5p inhibitor were protected from cartilage degradation and had reduced proteoglycan loss and reduced loss of articular chondrocyte cellularity compared with control mice. miR-218-5p is a novel inducer of cartilage destruction via modulation of PI3K/Akt/mTOR signaling. Inhibition of endogenous miR-218-5p expression/activity appears to be an attractive approach to OA treatment.

Association of receptor activator of nuclear factor-kappaB ligand (RANKL) gene polymorphisms with the susceptibility to ankylosing spondylitis: a case-control study.

OBJECTIVES: To investigate the association between receptor activator of nuclear factor-kappaB ligand (RANKL) gene polymorphisms and the susceptibility to ankylosing spondylitis (AS) in a Chinese Han population. METHODS: Three hundred and fifty-two AS patients and 299 age- and gender-matched controls were recruited in this study. Peripheral blood samples were collected from all the subjects and the genomic DNA was then extracted. Three single nucleotide polymorphisms (SNPs) of the RANKL gene (rs2277438, rs7984870 and rs9533156) were genotyped using the TaqMan assay. The frequencies of alleles and genotypes were compared between AS patients and normal controls. RESULTS: The distributions of genotype frequencies in rs2277438 were significantly different between AS patients and normal controls (P < 0.05). The frequency of G allele of SNP rs2277438 in AS patients was significantly higher than that in normal controls (P < 0.05). The frequencies of genotypes with G allele (GG and AG) were significantly higher in AS patients when compared with normal controls (OR = 1.573, 95 % CI 1.151-2.150, P < 0.05). Neither the genotype frequencies nor the allele frequencies of rs7984870 and rs9533156 were found to be significantly different between AS patients and normal controls (P > 0.05). CONCLUSIONS: The current study demonstrated that SNP rs2277438 of the RANKL gene was associated with the susceptibility of AS in a Chinese Han population. Genotypes with G allele (GG and AG) were identified as the risk factors for the occurrence of AS.

Lack of associations between two previously identified susceptible single nucleotide polymorphisms of interleukin-23 receptor gene and ankylosing spondylitis: a replication study in a Chinese Han population.

BACKGROUND: The human leukocyte antigen (HLA)-B27 gene is considered to be a major gene associated with predisposition to ankylosing spondylitis (AS); however, studies have demonstrated that non-HLA-B27 genes also contribute substantially to the susceptibility to AS. Two single nucleotide polymorphisms (SNPs), rs1004819 and rs10889677, of the interleukin-23 receptor (IL-23R) gene have been shown to be associated with AS susceptibility in European populations. However, ethnicity factors contribute to population splitting and genetic variation, and ethnic-specific genetic association studies are needed to validate these associations in patients from different ethnic backgrounds. This study therefore aimed to replicate the associations between these two SNPs and AS susceptibility in a Chinese Han population. METHODS: A total of 195 AS patients and 203 normal controls were recruited in this study. Two IL-23R gene SNPs, rs1004819 and rs10889677 were selected. Genotyping was performed in all subjects using the TaqMan probe method. Genotype and allele frequencies were compared between AS patients and normal controls by χ2 tests. RESULTS: There were no significant differences in either the genotype frequencies (TT 36.4%, TC 48.7% and CC 14.9% in AS patients; TT 35.0%, TC 50.0% and CC 15.0% in normal controls) or allele frequencies (T 60.8% and C 39.2% in AS patients; T 60.0% and C 40.0% in normal controls) of rs1004819 between AS patients and normal controls (P > 0.05). In addition, both the genotype frequencies (AA 51.3%, AC 43.1% and CC 5.6% in AS patients; AA 57.6%, AC 35.5% and CC 6.9% in normal controls) and allele frequencies (A 72.8% and C 27.2% in AS patients; A 75.4% and C 24.6% in normal controls) of rs10889677 were also comparable between AS patients and normal controls (P > 0.05). CONCLUSIONS: This study found no evidence for an association between either of the two previously identified AS-susceptibility IL-23R SNPs (rs1004819 and rs10889677) and onset of AS, indicating a possible difference in pathogenesis of AS between Chinese and European patients.

Anatomic relationship between superior mesenteric artery and aorta before and after surgical correction of thoracolumbar kyphosis.

STUDY DESIGN: A computed tomographic study. OBJECTIVE: To investigate the changed anatomic relationship between the superior mesenteric artery and the aorta in surgically treated ankylosing spondylitis (AS) patients with thoracolumbar kyphosis. SUMMARY OF BACKGROUND DATA: Superior mesenteric artery syndrome in kyphosis patients after surgery has been reported. To date, the changed aortomesenteric angle and distance in AS patients undergoing surgical correction of kyphosis have not yet been addressed. METHODS: Thirty-three AS patients with thoracolumbar kyphosis subjected to pedicle subtraction osteotomy were prospectively recruited. Radiographic measurements included global kyphosis (GK), thoracic kyphosis (TK), local kyphosis (LK), and lumbar lordosis. The aortomesenteric angle and distance were measured on both preoperative and postoperative computed tomography images. The height and weight of these patients were also recorded. RESULTS: The average height significantly increased from 159.7±5.1 cm before surgery to 167.2±5.3 cm after surgery (P<0.001), whereas the average weight changed from 59.3±6.8 kg to 59.5±6.9 kg (P>0.05). GK, TK, LK, and lumbar lordosis were corrected from 77.7, 40.2, 19.4, and 2.8 degrees before surgery to 37.1, 38.4, -21.9, and -32 degrees after surgery, respectively. All the changes of these parameters were found to be significantly different (P<0.001) except that of TK (P>0.05). With the correction of kyphosis, the aortomesenteric angle significantly decreased from 29.3 to 23.4 degrees (P<0.001), whereas the aortomesenteric distance significantly decreased from 25.7 to 20.4 mm (P<0.001). It is to be noted that the changes of GK, LK, and height were significantly correlated with the decrements of aortomesenteric angle and distance (P<0.05). CONCLUSIONS: The correlations of the surgical correction of kyphosis and the decreased aortomesenteric angle and distance in AS patients were quantitatively confirmed by our study. Spine surgeons should be aware of the potential risk for the development of superior mesenteric artery syndrome after kyphosis correction in AS patients.

An exon polymorphism of programmed cell death 1 gene is associated with both the susceptibility and thoracolumbar kyphosis severity of ankylosing spondylitis in a Chinese Han population.

BACKGROUND: Although human leukocyte antigen (HLA)-B27 gene is the major susceptible gene associated with ankylosing spondylitis (AS), it has been recognized that non-HLA-B27 genes also play key roles in the development of AS. The purpose of this study is to investigate whether a single nucleotide polymorphism (SNP) in the exon region of the programmed cell death 1 (PDCD-1) gene is associated with the susceptibility or the thoracolumbar kyphosis severity of AS in a Chinese Han population. METHODS: A total of 255 AS patients between January 2008 and October 2012 were recruited in this study. Two hundred and three healthy patients were recruited as normal controls. According to the severity of thoracolumbar kyphosis, the AS patients were further divided into group A (patients with kyphosis <70°, n = 135) and group B (patients with kyphosis ≥70°, n = 120). One exon polymorphism, rs2227982 (C/T) of PDCD-1 gene, was selected for analysis. Genotyping was performed by TaqMan probe assays in all the subjects. RESULTS: There were significant differences of genotype distributions of rs2227982 between AS patients and normal controls. The frequency of the T allele was significantly higher in AS patients when compared with normal controls. The frequency of the T allele in group B was significantly higher than that in group A. Carriage of the TT genotype increased the risk of severe thoracolumbar kyphosis 1.9-fold in AS patients. CONCLUSIONS: Our study confirms a significant association between the SNP rs2227982 of PDCD-1 gene and the susceptibility of AS in a Chinese Han population. Moreover, the TT genotype is suggested to be associated with the severity of thoracolumbar kyphosis secondary to AS.

[Osteotomy for severe thoracolumbar kyphosis in advanced ankylosing spondylitis: skipping two-level pedicle subtraction osteotomy].

OBJECTIVE: To explore the feasibility of single-stage skipping two-level pedicle subtraction osteotomy (PSO) for severe thoracolumbar kyphosis (Cobb > 100°) in advanced ankylosing spondylitis (AS). METHODS: Ten AS patients with thoracolumbar kyphosis undergoing skipping two-level PSO were retrospectively reviewed. The most frequent levels of osteotomy was L1 and L4 (n = 7), followed by T12 and L3 (n = 2) and L2 and L5 (n = 1). All patients were males with a mean age of 28.5 ± 9.1 years (range: 17 - 47). The pre- and post-operative values of thoracic kyphosis (TK), lumbar lordosis (LL), globe kyphosis (GK), local kyphosis of osteotomized vertebra (LK1, LK2) and sagittal imbalance (SVA) were measured. RESULTS: Significant differences were observed with respects to the improvements of LL, GK, LK1, LK2 and SVA (P < 0.01). LL, GK, LK1, LK2 and SVA improved from 41.9°, 113.4°, 40.5°, -0.3° and 25.2 cm preoperatively to -44.1°, 71.6°, 13.5°,-26.8° and 5.8 cm postoperatively respectively. The mean operative duration was 370 minutes (range: 290 - 420) and the estimated volume of blood loss 2600 ml (range: 1700 - 3800). Dural tear occurred intra-operatively in 1 patient. One had a transient brachial plexus paralysis and resolved after 1 week postoperatively. One had transient radiculopathy in right lower extremity and recovered completely 3 weeks postoperatively. CONCLUSION: As a safe and effective technique for correction of severe thoracolumbar kyphosis (Cobb > 100°) secondary to AS, single-stage skipping two-level PSO osteotomy can achieve larger correction and better sagittal alignment with a mean correction of 86°in terms of LL.

Isolated extradural tuberculous granuloma of the cervical spine: a case report.

INTRODUCTION: Isolated intraspinal extradural tuberculous granuloma (IETG) without radiological evidence of vertebral involvement is uncommon, especially rare in cervical spine. MATERIALS AND METHODS: We report a case of cervical IETG without bone involvement in a patient with neurological deficit. The patient suffered from progressive neurological dysfunction. MRI of cervical spine revealed an intraspinal extradural mass, and the spinal cord was edematous because of the compression. Thus C2-C4 laminectomy was performed and extradural mass was excised. RESULTS: The excised extradural mass was confirmed to be tuberculous granuloma through pathologic examination. Antituberculous drugs were administrated with a regular follow-up. Excellent clinical outcomes were achieved. CONCLUSIONS: The isolated IETG, although a rare entity, should be considered in the differential diagnosis of the intraspinal mass, especially in patients with spinal cord compression and a history of tuberculosis. If there is a progressing neurological deficit, a combination of surgical and anti-tuberculous treatment should be the optimal choice.

Pedicle subtraction osteotomy through pseudarthrosis to correct thoracolumbar kyphotic deformity in advanced ankylosing spondylitis.

INTRODUCTION: Surgical treatment is mandatory for spinal pseudarthrosis in advanced ankylosing spondylitis (AS) patients with painful sagittal deformity and/or neurological deficits. However, the most effective and safe surgical procedure for AS-related symptomatic thoracolumbar pseudarthrosis is still controversial. The purpose of this study is to explore the outcomes of pedicle subtraction osteotomy (PSO) at the level of pseudarthrotic lesion combined with supplemental anterior fusion for patients suffering from kyphotic pseudarthrosis in AS. MATERIALS AND METHODS: Seven AS patients with thoracolumbar pseudarthrosis and kyphotic deformity were reviewed. There were 6 males and 1 female with a mean age of 41.7 years. All patients had back pain. Imaging findings demonstrated 3-column extensive discovertebral destruction in all patients. The preoperative global kyphosis averaged 75° (range, 37°-114°) with the apex at the level of pseudarthrosis. Three patients had incomplete neurological deficits (Frankel D) preoperatively. All patients underwent PSO at the level of pseudarthrosis in the first stage followed by supplemental anterior fusion in the second stage. Radiographic and clinical outcomes were assessed with an average follow-up of 38 months (range, 24-59 months). The visual analogue scale (VAS) was compared before surgery and at the final follow-up. RESULTS: All patients showed significant pain relief postoperatively and were satisfied with the kyphosis correction as well. Solid bony fusion was shown at the final follow-up. Three patients with neurological deficits had complete recovery of neurological function. The global kyphosis was corrected from 75º to 30º, with a mean correction of 45º. The VAS showed significant improvement. No surgical complication was observed. CONCLUSION: PSO can be safely performed through the site of pseudarthrotic lesion in AS patients with pseudarthrosis and kyphotic deformity. After PSO, supplemental anterior fusion is sometimes necessary to support the anterior and middle column in a second stage if there is a bone defect in the osteotomy site.

Mutation analysis of MESP2, HES7 and DUSP6 gene exons in patients with congenital scoliosis.

MESP2, HES7 and DUSP6 genes have been proved to be involved in the etiopathogenesis of congenital scoliosis (CS) in animal embryo studies, however, whether this association was detected in human CS patients also remains unknown. One hundred sporadic and non-syndromic CS patients and 100 age-matched normal controls were included in this study. Mutation screening of gene exons were performed by DNA sequencing. However, no mutation or new single nucleotide polymorphism was found in the exons of MESP2, HES7 and DUSP6 genes in CS patients and normal controls. MESP2, HES7 and DUSP6 genes may not be involved in the etiopathogenesis of sporadic and non-syndromic CS in Chinese Han population.

Sirt6 attenuates chondrocyte senescence and osteoarthritis progression.

Sirt6 has been implicated as a key regulator in aging-related diseases, including osteoarthritis. However, its functional role and molecular mechanism in chondrocyte senescence and osteoarthritis pathophysiology remain largely undefined. Here we show that Sirt6 deficiency exaggerates chondrocyte senescence and osteoarthritis progression, whereas intra-articular injection of adenovirus-Sirt6 markedly attenuates surgical destabilization of medial meniscus-induced osteoarthritis. Mechanistically, Sirt6 can directly interact with STAT5 and deacetylate STAT5, thus inhibiting the IL-15/JAK3-induced STAT5 translocation from cytoplasm to nucleus, which inactivates IL-15/JAK3/STAT5 signaling. Mass spectrometry revealed that Sirt6 deacetylated conserved lysine 163 on STAT5. Mutation of lysine 163 to arginine in STAT5 abolished the regulatory effect of Sirt6. In vivo, specific ablation of Sirt6 in chondrocytes exacerbated osteoarthritis. Pharmacological activation of Sirt6 substantially alleviated chondrocyte senescence. Taken together, Sirt6 attenuates chondrocyte senescence by inhibiting IL-15/JAK3/STAT5 signaling. Targeting Sirt6 represents a promising new approach for osteoarthritis.

Tendon-to-Bone Healing in a Rat Extra-articular Bone Tunnel Model: A Comparison of Fresh Autologous Bone Marrow and Bone Marrow-Derived Mesenchymal Stem Cells.

BACKGROUND: Despite widespread acceptance of fresh autologous bone marrow (BM) for use in clinical practice, limited information exists to analyze if tendon-to-bone healing could be accelerated with local use of fresh autologous BM. PURPOSE: To investigate the effect of fresh autologous BM on tendon-to-bone healing with a novel rat model. STUDY DESIGN: Controlled laboratory study. METHODS: An extra-articular bone tunnel was created and filled with an autologous tendon graft in skeletally mature Sprague-Dawley rats (N = 60). They were then randomly divided into 3 groups: BM group (injection of fresh autologous BM into the tendon-bone interface, n = 20), BM-derived mesenchymal stem cell (BMSC) group (injection of allogenic cultured BMSCs, n = 20), and the control group (tendon-bone interface without injection of BM or BMSCs, n = 20). Biomechanical, histological, and immunohistochemical analyses were performed at 2 and 6 weeks after surgery. RESULTS: The BM group showed a relatively well-organized and dense connective tissue interface with better orientation of collagen fibers as compared with the BMSC group. At 2 weeks, the tendon-bone interface tissue thickness of the BMSC group was 140 ± 25 µm (mean ± SEM), which was significantly greater than the BM group (58 ± 15 µm). The BM group showed fewer M1 macrophages at the tendon-bone interface at 2 and 6 weeks (P < .001). In contrast, there were more M2 macrophages at the interface in the BM group 2 and 6 weeks postoperatively when compared with controls and the BMSC group (P < .001). Biomechanical tests revealed significantly higher stiffness in the BM group versus the control and BMSC groups at 2 and 6 weeks after surgery (P < .05). Load to failure showed similar trends to stiffness. CONCLUSION: These findings indicate that local delivery of fresh autologous BM enhances tendon-to-bone healing better than the alternative treatments in this study. This effect may be partially due to the observed modulation of inflammatory processes, especially in M2 macrophage polarization. CLINICAL RELEVANCE: Fresh autologous BM could be a treatment option for this disorder.

Preclinical development of a microRNA-based therapy for intervertebral disc degeneration.

Understanding the molecular mechanisms regulating the maintenance and destruction of intervertebral disc may lead to the development of new therapies for intervertebral disc degeneration (IDD). Here we present evidence from miRNA microarray analyses of clinical data sets along with in vitro and in vivo experiments that miR-141 is a key regulator of IDD. Gain- and loss-of-function studies show that miR-141 drives IDD by inducing nucleus pulposus (NP) apoptosis. Furthermore, miR-141 KO in mice attenuated spontaneous and surgically induced IDD. Mechanistically, miR-141 promotes IDD development by targeting and depleting SIRT1, a negative regulator of NF-κB pathway. Therapeutically, upregulation or downregulation of miR-141 by nanoparticle delivery in IDD model aggravated or alleviated experimental IDD, respectively. Our findings reveal a novel mechanism by which miR-141, in part, promotes IDD progression by interacting with SIRT1/NF-κB pathway. Blockade of miR-141 in vivo may serve as a potential therapeutic approach in the treatment of IDD.

Does the Position of the Aorta Change With the Altered Body Position in Ankylosing Spondylitis Patients With Thoracolumbar Kyphosis?: A Magnetic Resonance Imaging Investigation.

STUDY DESIGN: A prospective magnetic resonance imaging study. OBJECTIVE: To quantitatively explore the differences in the anatomic position of the aorta relative to the spine between supine and prone positions in ankylosing spondylitis (AS) patients with thoracolumbar kyphosis. SUMMARY OF BACKGROUND DATA: Aortic complications may occur during the lumbar spine osteotomy in correcting thoracolumbar kyphosis secondary to AS, and a clear understanding of the spatial relationship between the aorta and the vertebrae is essential to prevent these iatrogenic complications. However, previous anatomic study was performed with AS patients in the supine position, which was different from the prone position adopted in surgery. To date, no report has been published to investigate the mobility of the aorta relative to the vertebrae between supine and prone positions in AS patients with thoracolumbar kyphosis. MATERIALS AND METHODS: From March 2013 to September 2014, 22 AS patients (21 males, 1 female) with thoracolumbar kyphosis with a mean age of 30.7 years (range, 19-46 y) were recruited. Magnetic resonance imaging examinations from T9 to L3 in both the supine and prone positions were performed, and the left pedicle-aorta (LtP-Ao) angle and LtP-Ao distance were measured at each level. The differences of these parameters between the 2 positions were compared by the paired sample t test, and the relationships between the shifting of the aorta and the change of global kyphosis and lumbar lordosis were evaluated by the Pearson correlation coefficient. The level of significance (α) was set at 0.05. RESULTS: At T9-L3 levels, no significant difference was noted in LtP-Ao distances (43.78 vs. 44.42 mm; P=0.077) and LtP-Ao angles (0.82 vs. 0.22 degrees; P=0.053) between supine and prone positions. The correlation analysis also revealed no remarkable correlation between the change of LtP-Ao angle and increase of global kyphosis and lumbar lordosis in the prone position. CONCLUSIONS: There is no significant change of the relative positions between the aorta and the vertebrae at T9-L3 levels after the patient turned to a prone position, which implied that the mobility and range of motion of the aorta is limited in advanced stage of AS.

Identification of Serum miR-146a and miR-155 as Novel Noninvasive Complementary Biomarkers for Ankylosing Spondylitis.

STUDY DESIGN: Prospective serum microRNAs study. OBJECTIVE: To investigate differentially expressed serum microRNAs (miRNAs) between ankylosing spondylitis (AS) patients and controls, and to evaluate the potential of miRNAs as biomarkers for AS. SUMMARY OF BACKGROUND DATA: There is an urgent need to explore novel biomarkers with more high sensitivity and specificity for the diagnosis of AS, especially for early-stage AS. Recently, the discovery of miRNAs has paved a new avenue for the diagnosis of cancers and other diseases, However, the global serum miRNAs pattern in AS patients has never been determined. METHODS: An initial screening of miRNA expression by Solexa sequencing was performed using serum samples pooled from 10 AS patients and 10 controls, respectively. Subsequently, differential expression was validated using hydrolysis probe-based stem-loop quantitative reverse transcription polymerase chain reaction (qRT-PCR). Furthermore, AS patients were divided into group A (kyphosis <70°) and group B (kyphosis ≥70°). Disease activity and functional status were evaluated by Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) and the Bath Ankylosing Spondylitis Functional Index (BASFI), respectively. The areas under the receiver-operating characteristic (ROC) curves of identified miRNAs were analyzed. RESULTS: Nineteen serum miRNAs were differentially expressed in AS patients compared with controls. After qRT-PCR confirmation, miR-146a and miR-155 were significantly upregulated in AS patients. The areas under the ROC curves using miR-146a and miR-155 were 0.917 and 0.964, respectively. Importantly, the miR-155 expression level in group B was significantly higher than that in group A. In addition, significant correlation was observed between miR-155 expression level and BASDAI (r = 0.5, P < 0.01). CONCLUSION: Detection of serum miRNAs (miR-146a and miR-155) may serve as novel complementary biomarkers for AS. Moreover, the expression level of miR-155 is suggested to be associated with disease activity and the severity of thoracolumbar kyphosis secondary to AS. LEVEL OF EVIDENCE: NA.

Downregulation of microRNA-193a-3p is involved in invertebral disc degeneration by targeting MMP14.

UNLABELLED: Accumulating evidence suggests that microRNAs (miRNAs) play an important role in intervertebral disc degeneration (IDD), but the precise role of specific miRNAs involved in this disease remains elusive. The purpose of this study was to identify IDD-specific miRNAs, followed by functional validation of results. MiRNA expression profile was determined in nucleus pulposus (NP) tissues from patients with IDD and controls, employing Solexa sequencing and quantitative real-time PCR (qRT-PCR). Biological functions of differential expression miRNAs were further investigated in vitro and in vivo. Luciferase reporter assays and Western blotting were performed to determine miRNA targets. We identified 28 miRNAs that were differentially expressed in patients compared with controls. Following qRT-PCR confirmation, miR-193a-3p was significantly down-regulated in degenerative NP tissues. Moreover, its level was correlated with grade of disc degeneration. Through gain- and loss-of-function studies, miR-193a-3p was demonstrated to significantly promote type II collagen expression in NP cells. Knockdown of MMP14 induced effects on NP cells similar to those induced by miR-193a-3p. Bioinformatics target prediction identified MMP14 as a putative target of miR-193a-3p. Furthermore, luciferase reporter assays and Western blotting demonstrated that miR-193a-3p directly targets MMP14. MiR-193a-3p inhibited IDD in vitro and in vivo. The downregulation of miR-193a-3p induces the expression of MMP14, which promotes loss of type II collagen and thereby contributes to the development of human IDD. Our findings extend the role of miR-193a-3p in the pathogenesis of IDD and provide a potential novel therapeutic target for degenerative disc disease. KEY MESSAGES: Intervertebral disc degeneration (ICC)-specific miRNA profile generated by next generation sequencing. Downregulation of miR-193a-3p promoted loss of type II collagen by directly targeting MMP14 in IDD. miR-193a-3p inhibited IDD in vitro and in vivo. miR-193a-3p may be a promising candidate for prevention of degenerative disc disease.

Dysregulated miR-98 Contributes to Extracellular Matrix Degradation by Targeting IL-6/STAT3 Signaling Pathway in Human Intervertebral Disc Degeneration.

Intervertebral disc degeneration (IDD) is associated with dysregulated expression of microRNAs (miRNAs). However, the precise molecular mechanisms underlying this disorder remain unclear. Therefore, we tested the hypothesis that miRNAs modulate IDD through effects on the IL-6/STAT3 signaling pathway, a potential regulator of IDD. The miRNA expression profile was determined in nucleus pulposus (NP) tissues from patients with IDD and controls, employing miRNA microarray and quantitative real-time PCR (RT-qPCR). Biological functions of differential expression miRNAs were further investigated using immunofluorescent staining. Luciferase reporter assays and Western blotting were performed to determine miRNA targets. We identified 41 miRNAs that were differentially expressed in patients compared with controls. Following RT-qPCR confirmation, miR-98 was significantly downregulated in degenerative NP tissues. Moreover, its level was inversely correlated with grade of disc degeneration. Through gain-of-function and loss-of-function studies, miR-98 was shown to significantly promote type II collagen expression in NP cells. Interleukin-6 (IL-6) was identified as a target of miR-98. Knockdown of IL-6 induced effects on NP cells similar to those induced by miR-98. In contrast, IL-6 treatment abrogated the effects induced by miR-98 upregulation. Moreover, miR-98 dramatically suppressed expression of STAT3 target gene, MMP2. IL-6 treatment antagonized this effect, whereas knockdown of IL-6 by IL-6 short hairpin RNA (shIL-6) induced inhibitory effects on the expression of p-STAT3 and its main target genes, similar to miR-98. The mRNA level of IL-6 was inversely correlated with that of miR-98 in degenerative NP tissues. These results suggest the downregulation of miR-98 could promote IDD through the IL-6/STAT3 signaling pathway. Our findings also highlight miR-98 as a novel hopeful therapeutic target for IDD.

Is There any Correlation Between Pathological Profile of Facet Joints and Clinical Feature in Patients With Thoracolumbar Kyphosis Secondary to Ankylosing Spondylitis?: An Immunohistochemical Investigation.

STUDY DESIGN: An immunohistochemical analysis. OBJECTIVE: The aim of this study was to systematically and extensively evaluate the immunopathology of the facet joints in patients with thoracolumbar kyphosis secondary to ankylosing spondylitis (AS). SUMMARY OF BACKGROUND DATA: The facet joints may be predominantly involved in the process of spinal inflammation in AS. Thus, a detailed investigation of the immunopathology at sites of facet joints is of crucial importance in understanding the pathogenesis of AS. METHODS: The facet joints were obtained from 30 AS patients and 23 age- and gender-matched controls (patients with fresh thoracolumbar fracture). The facet joints were assessed immunohistochemically by analyzing the number of infiltrating T cells (CD3, CD4, CD8), B cells (CD20), microvessel density (CD34), osteoblasts (CD56), bone marrow macrophages (CD68), and osteoclasts (CD68) per high-power field (hpf). According to the presence or absence of persistent inflammation, AS patients were divided into 2 groups: A (patients with persistent inflammation) and B (patients without persistent inflammation). Lumbar spinal mobility was assessed using the modified Schober index (MSI). RESULTS: Two or more CD3+ T cell aggregates were found in the facet joints from 18 of 30 AS patients, whereas 1 CD3+ T cell aggregate was noted in 5 of 23 patients with thoracolumbar fracture. The levels of T cells (CD4+ and CD8+), CD20+B cells, CD56+ osteoblasts, and CD34+ microvessel density were significantly higher in AS patients than in the controls (all P < 0.01). Notably, the MSI score in group A was significantly higher than that in group B (P < 0.01). CONCLUSION: Active spinal inflammation is frequently observed in AS patients with thoracolumbar kyphosis. In addition, persistent inflammation in facet joints may further contribute to the loss of spinal mobility in the later stages of AS. These findings indicate that careful monitoring of disease activity is mandatory for AS patients in its advanced stage. LEVEL OF EVIDENCE: 4.