Depletion of circRNA circ_CDK14 inhibits osteosarcoma progression by regulating the miR-520a-3p/GAB1 axis.

Osteosarcoma (OS) is a lethal bone malignancy. Circular RNAs (circRNAs) have emerged as important regulators of OS development. CircRNA cyclin dependent kinase 14 (circ_CDK14) was reported to be a potential oncogene in OS. However, the mechanistic pathway by which circ_CDK14 functions in OS is largely unknown. The relative expression of circ_CDK14, microRNA (miR)-520a-3p, and GRB2 Associated Binding Protein 1 (GAB1) was evaluated by quantitative real-time PCR and western blot assays. Flow cytometry was employed to monitor cell cycle distribution and apoptosis. Methyl thiazolyl tetrazolium (MTT) and colony formation assays were performed to assess cell viability and colony formation ability, respectively. Western blot assay was also used to detect the expression of apoptosis-related proteins. Transwell assay was carried out to monitor cell migration and invasion. Additionally, the target association between miR-520a-3p and circ_CDK14 or GAB1 was confirmed by a dual-luciferase reporter assay. Xenograft assay was applied to investigate the role of circ_CDK14 in vivo. Circ_CDK14 and GAB1 expression was upregulated, while miR-520a-3p was downregulated in OS tissues and cells. Circ_CDK14 depletion hindered OS cell proliferation, metastasis, and tumorigenesis while facilitated apoptosis, which were all ameliorated by miR-520a-3p inhibition. Circ_CDK14 could sponge miR-520a-3p. miR-520a-3p targeted GAB1 to repress OS cell proliferation and metastasis. Circ_CDK14 knockdown blocked OS tumor growth in vivo. Circ_CDK14 might positively affect OS development by modulating the miR-520a-3p/GAB1 axis.

Roles of miR-182 in Intervertebral Disc Degeneration and its Potential Prognostic Value.

BACKGROUND: MicroRNAs were reported to be involved in the progression of intervertebral disc degeneration (IDD). This study focused on the potential prognostic value and the underlying mechanism of miR-182 in IDD. METHODS: The expression level of miR-182 in plasma samples from 60 IDD patients and 60 healthy controls were examined in the present study. Then, the relationship between miR-182 expression and clinical features of IDD patients was analyzed. Moreover, the nucleus pulposus (NP) cells were cultured and transfected with either miR-182 inhibitor, mimics, or NC to explore the effects of miR-182 on cell proliferation and apoptosis. Furthermore, expression levels of proliferation and apoptosis-related proteins Bcl-2, Bax, and Caspase-3 were also evaluated. RESULTS: The expression level of miR-182 was dramatically increased in plasma samples of IDD patients compared with the controls. Moreover, ROC analysis indicated that miR-182 was a feasible diagnostic indicator for the diagnosis of IDD. According to the Japanese Orthopaedic Association (JOA) score, the prognosis of patients with the lower expression levels of miR-182 was better than for those with the higher expression levels of miR-182 in IDD. Furthermore, the miR-182 inhibitor significantly increased the proliferation, decreased the apoptosis of human NP cells, and altered the expression of proliferation and apoptosis-related proteins Bcl-2, Bax, and Caspase-3. On the contrary, miR-182 mimics notably inhibited proliferation but promoted apoptosis of human NP cells and increased Bax and Caspase-3 expressions while reducing the Bcl-2 level. CONCLUSIONS: miR-182 was negatively correlated with the prognosis of the IDD patients and affected the proliferation and apoptosis of NP cells in vitro.

Upregulation of suppressor of cytokine signaling 3 ameliorates spinal degenerative disease in adolescents by mediating leptin and tumor necrosis factor-α levels.

Spinal degenerative changes may occur following the rapid growth observed in adolescents, causing a reduced quality of life. The suppressor of cytokine signaling (SOCS) is involved in various degenerative diseases. The current study recruited adolescents with spinal degenerative disease (SDD) to identify the effect of SOCS-3 on leptin and tumor necrosis factor-α (TNF-α) levels in this disorder. From January 2010 to January 2016, 120 adolescents (aged 14 to 25) were enrolled in the current study, with 68 diagnosed with SDD and the remaining 52 treated as controls. Nucleus pulposus cells (NPCs) were extracted and cultured in vitro. TNF-α levels in NPCs were determined using flow cytometry. Degenerative NPCs were then transfected with pCR3.1-SOCS-3 and ELISA was performed to determined TNF-α and leptin levels. RT-qPCR was performed to measure the mRNA level of SOCS-3 and leptin in NPCs and western blotting was utilized to detect the protein level of leptin and the extent of leptin receptor phosphorylation. The results revealed that TNF-α levels in degenerative NPCs were higher than those in normal NPCs. The overexpression of SOCS-3 reduced levels of TNF-α and leptin in degenerative NPCs. In addition, the upregulation of leptin increased SOCS-3 levels in a concentration-dependent manner. Furthermore, the expression of the leptin receptor and phosphorylated leptin receptor gradually decreased with increasing leptin concentrations and the level of phosphorylated leptin receptor negatively correlated with SOCS-3 expression. The inductive effect of leptin on the level of SOCS-3 and the inhibitory effect of SOCS-3 on the activity of leptin were identified. The current study demonstrated that SOCS-3 reduces leptin and TNF-α levels in degenerative NPCs from adolescents, indicating its potential role in the development of novel SDD therapies.

Thermal pretreatment promotes the protective effect of HSP70 against tendon adhesion in tendon healing by increasing HSP70 expression.

Tendon adhesion is a substantial challenge for tendon repair. Thermal pretreatment (TP) may decrease inflammation by upregulating heat shock proteins (HSPs). The present study intends to identify the function that TP serves when combined with HSP70 overexpression in tendon healing and adhesion in rats. Sprague­Dawley male rats were used to establish a surgically ablative tendon postoperative suture model, and the positive expression of the HSP70 protein was measured using immunohistochemistry. Changes to the blood vessels and collagenous fiber, in addition to the maximum tensile strength and the tendon sliding distance, were detected under a microscope. Finally, HSP70, tumor growth factor ß (TGF­ß), and insulin­like growth factor 1 (IGF­1) mRNA and protein levels were all determined by employing reverse transcription­quantitative polymerase chain reaction and western blot analysis methods. The positive expression of the HSP70 protein increased following TP. Furthermore, TP reduced the infiltration of inflammatory cells and improved the collagenous arrangement, accompanied by an increased maximum tensile force and tendon gliding distance following surgery. In addition, TP increased the mRNA and protein expression levels of HSP70, TGF­ß and IGF­1. Altogether, TP increases HSP70 expression, thereby reducing postoperative traumatic inflammation and establishing tendon adhesion and promoting tendon healing. Thus, TP may be a potential strategy for the treatment of tendon adhesion.

Down-regulation of long non-coding RNA ITGB2-AS1 inhibits osteosarcoma proliferation and metastasis by repressing Wnt/ß-catenin signalling and predicts favourable prognosis.

Osteosarcoma is the most common and highly aggressive bone neoplasm often occurs among adolescents and young people. In despite of the major advances in the treatment, the overall survival of osteosarcoma patients remains poor. Long non-coding RNAs (lncRNAs) have been identified as key regulators involved in tumorigenesis and progression of osteosarcoma. However, the exact roles and mechanisms of lncRNAs in osteosarcoma remain unclear. In this study, the functions and underlying molecular mechanisms of a novel lncRNA, ITGB2 antisense RNA1 (ITGB2-AS1) were investigated in osteosarcoma. The expression levels of ITGB2-AS1 were up-regulated in osteosarcoma tissue samples and cell lines determined by qRT-PCR assay. Osteosarcoma patients with high ITGB2-AS1 expression had a significantly poor prognosis. In addition, knockdown of ITGB2-AS1 inhibited the proliferation and induced apoptosis of osteosarcoma cells using MTT assay and flow cytometry detection. Furthermore, wound healing and transwell invasion assay revealed that depletion of ITGB2-AS1 suppressed the migration and invasion abilities of osteosarcoma cells. Molecular mechanism study indicated that ITGB2-AS1 inhibition impaired Wnt/ß-catenin signalling pathway in osteosarcoma cells. Taken together, our study suggested that ITGB2-AS1 played critical roles in the development and progression of osteosarcoma via Wnt/ß-catenin signalling, indicating that ITGB2-AS1 might be a valuable diagnostic biomarker and potential anticancer therapeutic target for osteosarcoma.

Dynamic and distinct histone modifications of osteogenic genes during osteogenic differentiation.

Many skeletal diseases have common pathological phenotype of defective osteogenesis of bone marrow stromal cells (BMSCs), in which histone modifications play an important role. However, few studies have examined the dynamics of distinct histone modifications during osteogenesis. In this study, we examined the dynamics of H3K9/K14 and H4K12 acetylation; H3K4 mono-, di- and tri-methylation; H3K9 di-methylation and H3K27 tri-methylation in osteogenic genes, runt-related transcription factor 2 (Runx2), osterix (Osx), alkaline phosphatase, bone sialoprotein and osteocalcin, during C3H10T1/2 osteogenesis. H3 and H4 acetylation and H3K4 di-methylation were elevated, and H3K9 di-methylation and H3K27 tri-methylation were reduced in osteogenic genes during C3H10T1/2 osteogenesis. C3H10T1/2 osteogenesis could be modulated by altering the patterns of H3 and H4 acetylation and H3K27 tri-methylation. In a glucocorticoid-induced osteoporosis mouse model, we observed the attenuation of osteogenic potential of osteoporotic BMSCs in parallel with H3 and H4 hypo-acetylation and H3K27 hyper-tri-methylation in Runx2 and Osx genes. When H3 and H4 acetylation was elevated, and H3K27 tri-methylation was reduced, the attenuated osteogenic potential of osteoporotic BMSCs was rescued effectively. These observations provide a deeper insight into the mechanisms of osteogenic differentiation and the pathophysiology of osteoporosis and can be used to design new drugs and develop new therapeutic methods to treat skeletal diseases.