MicroRNA-124 suppresses growth and aggressiveness of osteosarcoma and inhibits TGF-ß-mediated AKT/GSK-3ß/SNAIL-1 signaling.

Osteosarcoma is one of the most common malignant tumors in adolescent populations and the prognosis remains incompletely understand. Previous reports have demonstrated that microRNA­124 (miR­124) has inhibitory effects on various human malignancies and is associated with tumor progression. However, the clinical significance and potential mechanisms of miR­124 in the progression of osteosarcoma is not clearly understood. In this study, the potential molecular mechanism of miR­124 in osteosarcoma tumorigenesis, growth and aggressiveness was investigated. The growth, proliferation, apoptosis, migration and invasion of osteosarcoma cells were investigated following miR­124 transfection were determined by colony formation assay, western blotting, immunofluorescence, migration/invasion assays and reverse transcription­quantitative polymerase chain reaction. In vivo anti­cancer effects of miR­124 were analyzed by a tumor growth assay, immunohistochemistry and survival rate observations. The results demonstrated that miR­124 transfection significantly decreased integrin expression in osteosarcoma cells, and further inhibited growth, proliferation, migration and invasion of osteosarcoma cells. Flow cytometry assays indicated that miR­124 transfection attenuated apoptosis resistance of osteosarcoma to tunicamycin, potentially via the downregulation of P53 and Bcl­2 apoptosis regulator expression. Mechanistic assays demonstrated that miR­124 transfection suppressed TGF­ß expression in osteosarcoma. An animal study revealed that tumor growth was reduced in tumor cells transfected with miR­124 compared with control cells, and the survival rate was prolonged in mice with miR­124 transfected xenografts compared with control tumors. In conclusion, these results indicate that miR­124 transection inhibits the growth and aggressive of osteosarcoma, potentially via suppression of TGF­ß­mediated AKT/GSK­3ß/snail family transcriptional repressor 1 (SNAIL­1) signaling, suggesting miR­124 may be a potential anti­cancer agent/target for osteosarcoma therapy.

MicroRNA-300 Regulates the Ubiquitination of PTEN through the CRL4BDCAF13 E3 Ligase in Osteosarcoma Cells.

Cullins, critical members of the cullin-RING ubiquitin ligases (CRLs), are often aberrantly expressed in different cancers. However, the underlying mechanisms regarding aberrant expression of these cullins and the specific substrates of CRLs in different cancers are mostly unknown. Here, we demonstrate that overexpressed CUL4B in human osteosarcoma cells forms an E3 complex with DNA damage binding protein 1 (DDB1) and DDB1- and CUL4-associated factor 13 (DCAF13). In vitro and in vivo analyses indicated that the CRL4BDCAF13 E3 ligase specifically recognized the tumor suppressor PTEN (phosphatase and tensin homolog deleted on chromosome 10) for degradation, and disruption of this E3 ligase resulted in PTEN accumulation. Further analyses indicated that miR-300 directly targeted the 3' UTR of CUL4B, and DNA hypermethylation of a CpG island in the miR-300 promoter region contributed to the downregulation of miR-300. Interestingly, ectopic expression of miR-300 or treatment with 5-AZA-2'-deoxycytidine, a DNA methylation inhibitor, decreased the stability of CRL4BDCAF13 E3 ligase and reduced PTEN ubiquitination. By applying in vitro screening to identify small molecules that specifically inhibit CUL4B-DDB1 interaction, we found that TSC01131 could greatly inhibit osteosarcoma cell growth and could disrupt the stability of the CRL4BDCAF13 E3 ligase. Collectively, our findings shed new light on the molecular mechanism of CUL4B function and might also provide a new avenue for osteosarcoma therapy.

Activation of TNF-α/NF-κB axis enhances CRL4BDCAF11 E3 ligase activity and regulates cell cycle progression in human osteosarcoma cells.

Cullin 4B, a member of the Cullins, which serve as scaffolds to facilitate the assembly of E3 ligase complexes, is aberrantly expressed in many cancers, including osteosarcoma. Recently, we observed that CUL4B forms the CRL4BDCAF11 E3 ligase, which specifically ubiquitinates and degrades the cyclin-dependent kinase (CDK) inhibitor p21Cip1 in human osteosarcoma cells. However, the underlying mechanisms regarding the aberrant expression of CUL4B and the upstream members of this signaling pathway are mostly unknown. In this study, we demonstrate that nuclear factor kappaB (NF-κB) is a direct modulator of CUL4B expression. The CUL4B promoter is responsive to several NF-κB subunits, including RelA, RelB, and c-Rel, but not to p50 or p52. Additional studies reveal that the tumor necrosis factor alpha (TNF-α)/NF-κB axis pathway is activated in human osteosarcoma cells. This activation causes both CUL4B and NF-κB subunits to become abundant in the nucleus of human osteosarcoma cells. The down-regulation of individual genes, including TNFR1, RelA, RelB, c-Rel, and CUL4B, or pairs of them, including TNFR1 + RelA, TNFR1 + RelB, TNFR1 + c-Rel, and RelA+CUL4B, has similar effects on cell growth inhibition, colony formation, cell invasion, and in vivo tumor formation, whereas the overexpression of CUL4B in these knockdown cells significantly reverses their phenotypes. The inhibition of the TNF-α/NF-κB pathway greatly attenuates CRL4BDCAF11 E3 ligase activity and causes the accumulation of p21Cip1 , thereby leading to cell cycle arrest at the S phase. Taken together, our results support a model in which the activation of the TNF-α/NF-κB axis contributes to an increase in CRL4BDCAF11 activity and a decrease in p21Cip1 protein levels, thereby controlling cell cycle progression in human osteosarcoma cells.

Autophagic degradation of FOXO3a represses the expression of PUMA to block cell apoptosis in cisplatin-resistant osteosarcoma cells.

Autophagy and apoptosis are the two major modes of cell death, and autophagy usually inhibits apoptosis. The current understanding has shown that there is a complex crosstalk between the components of these two pathways. Here, we describe a transcriptional mechanism that links autophagy to apoptosis. We show that the cisplatin-resistant MG63-R12 and U2OS-R5 osteosarcoma sublines, in comparison to their parental MG63 and U2OS cells, respectively, exhibit increased autophagy but decreased apoptosis levels after treatment with cisplatin. We then used a microarray assay to examine the gene expression changes in these two cisplatin-resistant sublines and found that the expression of the transcription factor FOXO3a was dramatically decreased. Pharmacological treatment with either 3-methyladenine to inhibit autophagy or with rapamycin to activate autophagy in these two cisplatin-resistant sublines resulted in the accumulation or degradation of FOXO3a, respectively. Ectopic expression of FOXO3a in MG63-R12 and U2OS-R5 cells significantly enhanced cell sensitivity to cisplatin through a mechanism in which FOXO3a directly binds to the PUMA promoter and activates its expression, as well as its downstream event, the intrinsic apoptosis pathway. Importantly, this overexpression resulted in tumor growth inhibition in vivo. In conclusion, our results provide new insights into the molecular link between autophagy and apoptosis that involves a FOXO3a-mediated transcriptional mechanism. Importantly, our results may facilitate the development of therapeutic strategies for osteosarcoma patients who have become resistant to cisplatin therapy.

Leptin differentially regulates chondrogenesis in mouse vertebral and tibial growth plates.

BACKGROUND: Leptin plays an important role in mediating chondrogenesis of limb growth plate. Previous studies suggest that bone structures and development of spine and limb are different. The expression of Ob-Rb, the gene that encodes leptin receptors, is vertebral and appendicular region-specific, suggesting the regulation of leptin on VGP and TGP chondrogenesis may be very different. The aim of the present study was to investigate the differential regulation of leptin on the chondrogenesis of vertebral growth plate (VGP) and tibial growth plate (TGP). METHODS: We compared the VGP and TGP from wild type (C57BL/6) and leptin-deficient (ob/ob) mice. We then generated primary cultures of TGP and VGP chondrocytes. By treating the primary cells with different concentrations of leptin in vitro, we analyzed proliferation and apoptosis of the primary chondrocytes from TGP and VGP. We further measured expression of chondrogenic-related genes in these cells that had been incubated with different doses of leptin. RESULTS: Leptin-deficient mice of 8-week-old had shorter tibial and longer vertebral lengths than the wide type mice. Disturbed columnar structure was observed for TGP but not for VGP. In primary chondrocyte cultures, leptin inhibited VGP chondrocyte proliferation but promoted their apoptosis. Collagen IIA and aggrecan mRNA, and the protein levels of proliferation- and chondrogenesis-related markers, including PCNA, Sox9, and Smad4, were downregulated by leptin in a dose-dependent manner. In contrast, leptin stimulated the proliferation and chondrogenic differentiation of TGP chondrocytes at physiological levels (i.e., 10 and 50 ng/mL) but not at high levels (i.e., 100 and 1000 ng/mL). CONCLUSION: Leptin exerts a stimulatory effect on the proliferation and chondrogenic differentiation of the long bone growth plate but an inhibitory effect on the spine growth plate. The ongoing study will shed light on the regulatory mechanisms of leptin in bone development and metabolism.

Correlation of the Features of the Lumbar Multifidus Muscle With Facet Joint Osteoarthritis.

Facet joint osteoarthritis is considered a consequence of the aging process; however, there is evidence that it may be associated with degenerative changes of other structures. The goal of this study was to investigate the correlation between lumbar multifidus muscle features and facet joint osteoarthritis. This retrospective study included 160 patients who had acute or chronic low back pain and were diagnosed with facet joint osteoarthritis on computed tomography scan. Morphometric parameters, including cross-sectional area, muscle-fat index, and percentage of bilateral multifidus asymmetry at L3-L4, L4-L5, and L5-S1, were evaluated with T2-weighted magnetic resonance imaging. Patients with facet joint osteoarthritis had a smaller cross-sectional area and a higher muscle-fat index than those without facet joint osteoarthritis (P<.001). In multivariate regression analysis, older age and higher muscle-fat index were independently associated with facet joint osteoarthritis at all 3 spinal levels (P<.001). Smaller cross-sectional area was independently associated with facet joint osteoarthritis only at L4-L5 (P=.005). Asymmetry of the bilateral multifidus cross-sectional area was independently associated with facet joint osteoarthritis at L5-S1 (P=.009), but did not seem to be responsible for asymmetric degeneration of the bilateral facet joints. A higher multifidus muscle-fat index was independently associated with facet joint osteoarthritis, and bilateral multifidus size asymmetry was associated with the development of facet joint osteoarthritis at L5-S1. It seems more accurate to consider facet joint osteoarthritis a failure of the whole joint structure, including the paraspinal musculature, rather than simply a failure of the facet joint cartilage. [Orthopedics. 2017; 40(5):e793-e800.].

CRL4BDCAF11 E3 ligase targets p21 for degradation to control cell cycle progression in human osteosarcoma cells.

Cell cycle progression in mammals is strictly controlled by a number of cyclin-dependent kinases (CDKs) and CDK inhibitors (CKIs), the expression of which is often dysregulated in cancer cells. Our previous work revealed that Cullin 4B (CUL4B), a critical component of the Cullin4B-RING E3 ligase complex (CRL4B), is overexpressed in human osteosarcoma cells through an unknown mechanism. Here, we demonstrated that CUL4B forms an E3 ligase with RBX1 (RING-box 1), DDB1 (DNA damage binding protein 1), and DCAF11 (DDB1 and CUL4 associated factor 11) in human osteosarcoma cells. In vitro and in vivo ubiquitination analyses indicated that CRL4BDCAF11 E3 ligase was able to specifically ubiquitinate a CDK inhibitor-p21Cip1 at K16, K154, K161 and K163 but not at K75 and K141. Knocking down any component of the CRL4BDCAF11 complex, including CUL4B, DDB1 or DCAF11, using short hairpin RNAs (shRNAs) attenuated the ubiquitination level of p21Cip1, inhibited osteosarcoma cell proliferation, led to cell cycle arrest at S phase, and decreased colony formation rate. Taken together, our data suggest that the CRL4BDCAF11 complex represents a unique E3 ligase that promotes the ubiquitination of p21Cip1 and regulates cell cycle progression in human osteosarcoma cells.