mir-21-5p inhibits the progression of human chondrosarcoma by regulating CCR7/STAT3/NF-κB pathway.

Purpose: MicroRNAs (miRNAs or miRs) play an important role in the initiation and development of chondrosarcoma (CS). However, the role of miR-21-5p in CS progression and its underlying molecular mechanisms remains unclear.Materials and Methods: miR-21-5p expression was measured by qRT-PCR. Cell proliferation, migration, and invasion were detected by CCK-8 and Transwell assay. Dual-luciferase reporter assay was used to validate the target of miR-21-5p. Western blot was applied to explore the expressions of CCR7 and EMT biomarkers. Then, the xenograft model was established to confirm the effects of miR-21-5p.Results: miR-21-5p was significantly downregulated in CS tissues and cells and negatively correlated with grade, recurrence, and 5-year overall survival. In vitro, miR-21-5p caused G0/G1 cell cycle arrest and induced apoptosis by decreasing cyclin D1 expression and Bcl-2/Bax ratio. miR-21-5p suppressed cell migration and invasion of CS cells by inhibiting epithelial-mesenchymal transition (EMT). In vivo, miR-21-5p inhibited xenograft tumor formation of SW1353 cells. Mechanistically, miR-21-5p targeted the 3'-UTR of C-C chemokine receptor 7 (CCR7) mRNA to inhibit its expression. Overexpression of CCR7 reversed the inhibitory effects of miR-21-5p on CS cell behaviors. However, the silencing of CCR7 enhanced the inhibitory effects of miR-21-5p on CS cells. Besides, the overexpression of miR-21-5p or silencing of CCR7 obviously reduced the expression levels of p-STAT3, p-p56, and p-IκBα.Conclusion: miR-21-5p targeted CCR7 expression to inhibit the STAT3 and NF-κB signaling, thereby suppressing cell proliferation, migration, invasion, and EMT in CS cells, which might be a novel mechanistic study for CS therapy.Abbreviations: 3'-UTR: 3'-untranslated region; CCR7: C-C chemokine receptor type 7; CS: chondrosarcoma; DMEM: dulbecco's modified eagle's medium; EMT: epithelial-mesenchymal transition; HEK-293T: human embryonic kidney-293T; miR-21-5p: microRNA-21-5p; miR-NC: negative control miRNA; SD: standard deviation; si-CCR7: CCR7 siRNAs.

Effect of ß­ecdysterone on glucocorticoid­induced apoptosis and autophagy in osteoblasts.

The aim of the present study was to investigate the effect of glucocorticoids in osteoblasts and to examine the role of ß­ecdysterone in the pathogenesis of glucocorticoid­induced osteoporosis. Osteoblasts were induced from bone marrow mesenchymal stem cells, which were isolated from C57BL/6 mice. Cell viability and apoptosis of osteoblasts were measured by Cell Counting Kit­8 and flow cytometry analysis, respectively. The expression of related genes and proteins was measured by reverse transcription quantitative polymerase chain reaction and western blot analysis respectively. Dose­dependent decreases in the cell proliferation and differentiation were observed in dexamethasone (Dex)­treated osteoblasts, evidenced by downregulation in the activity of alkaline phosphatasedecreased expression levels of Runt­related transcription factor 2 and osteocalcin, and upregulated expression of RANK ligand. Dex also induced apoptosis and inhibited autophagy of osteoblasts, evidenced by upregulated B­cell lymphoma 2 (Bcl­2)­associated X protein/Bcl­2 ratio and the activation of mammalian target of rapamycin (mTOR), and decreased expression levels of Beclin­1, autophagy protein 5 and microtubule­associated protein 1 light chain 3 II. The effects on cell proliferation, apoptosis and autophagy induced by Dex were reversed by ß­ecdysterone in a dose­dependent manner. Therefore, ß­ecdysterone may be a promising candidate drug for the treatment of osteoporosis through inducing osteoblast autophagic activity by inactivating mTOR.

Iron/ABNO-Catalyzed Aerobic Oxidation of Alcohols to Aldehydes and Ketones under Ambient Atmosphere.

We report a new Fe(NO3)3·9H2O/9-azabicyclo[3.3.1]nonan-N-oxyl catalyst system that enables efficient aerobic oxidation of a broad range of primary and secondary alcohols to the corresponding aldehydes and ketones at room temperature with ambient air as the oxidant. The catalyst system exhibits excellent activity and selectivity for primary aliphatic alcohol oxidation. This procedure can also be scaled up. Kinetic analysis demonstrates that C-H bond cleavage is the rate-determining step and that cationic species are involved in the reaction.

A porphyrin-inspired iron catalyst for asymmetric epoxidation of electron-deficient olefins.

An in situ formed porphyrin-inspired iron complex that catalyzes asymmetric epoxidation of di- and trisubstituted enones is described. The reaction provides highly enantioenriched α,ß-epoxyketones (up to 99% ee). The practical utility of the new catalyst system is demonstrated by the gram-scale synthesis of optically pure epoxide. Hammett analysis suggests that the transition state of the reaction is electron-demanding and the active oxidant is electrophilic.

Slug signaling is up-regulated by CCL21/CCR7 [corrected] to induce EMT in human chondrosarcoma.

In recent decades, the CXC chemokine receptor 7 (CCR7) [corrected] and its ligand CCL21 have been extensively reported to be associated with tumorigenesis. Meanwhile, Slug signaling induces the epithelial-mesenchymal transition (EMT) process in chondrosarcoma development. In the present study, we explored the functions of CCL21/CCR7 [corrected] in Slug-mediated EMT in the chondrosarcoma. We analyzed protein expression of CCR7 [corrected] and Slug in human chondrosarcoma samples. Effects of CCR7 [corrected] on chondrosarcoma cells were assessed by in vitro assays. Additionally, CCR7 [corrected] pathways were further investigated by pharmacological and genetic approaches. We found that the altered CCR7 [corrected] (81.7 %) and Slug (85.0 %) expression in human chondrosarcoma tissues were significantly associated with grade, recurrence, and 5-year overall survival. According to in vitro assays, CCL21 stimulation induced the expression of phosph-ERK, phosph-AKT, Slug and N-cadherin in SW1353 cells, while the expression of E-cadherin was down-regulated. Furthermore, Slug signaling modulated E- to N-cadherin switch, which was influenced by the kinase inhibitor PD98059 and LY294002. In addition, the genetic silencing of Slug inhibited the capacity of migration and invasion of SW1353 cells. In conclusion, CCL21/CCR7 [corrected] pathway activates ERK and PI3K/AKT signallings to up-regulate Slug pathway, leading to the occurrence of EMT process in human chondrosarcoma. This study lays a new foundation for molecule-targeted therapy of human chondrosarcoma.

Inhibitory effects of pharmacological doses of dexamethasone on mineralization of mesenchymal progenitor cells in vitro.

Dexamethasone (Dex), a synthetic glucocorticoid, has a clinical adverse effect on bone acquisition and metabolism at pharmacological doses. To investigate the underlying mechanisms of Dex induced bone loss, we employed calvaria derived mesenchymal progenitor cells (MPCs) to examine the effects of Dex on their osteoblast lineage commitment and mineralization function. MPCs were cultured up to 28 days in the presence or absence of pharmacological doses of Dex. Alkaline phosphatase (ALP) and von Kossa histochemical staining showed that Dex decreased ALP activity and mineralized nodule formation. In addition, Dex treatment led to inhibition of cell proliferation and a decrease of cell numbers as assessed by BrdU incorporation and MTT methods, while it increased apoptosis as shown by flow cytometry of annexin V-stained cells. These effects were associated with a marked reduction of secreted IGF-I levels as indicated by ELISA quantification, raising the possibility that Dex decreased proliferation and promoted apoptosis of MPCs through the inhibition of IGF-I secretion. To further define the effect of Dex on osteoblast lineage commitment, Runx2 and Osx, the key transcription regulators of osteogenesis, were determined at both mRNA and protein levels. Interestingly, no effects were observed on mRNA and protein expression of Osx, while the mRNA and protein levels of Runx2 were inhibited by Dex treatment. Taken together, the inhibition of the expression of IGF-I and Runx2 by Dex in this in vitro system may account for the impaired MCP proliferation, osteoblastic differentiation and mineralized matrix deposition. These findings and the in vitro MCP system developed will facilitate further mechanistic studies of glucocorticoid induced bone loss.

OIC-A006 promotes osteogenesis in vitro and in vivo.

Bone morphogenesis proteins (BMPs) are one of the potent bone-forming factors. However, the safety, utility, and cost effectiveness of BMPs must be considered. Nowadays, there has been substantial interest in developing a chemical compound that safely promotes bone formation and facilitates fracture repair. Based on previous research with high throughout screening assay, we found one potent osteogenic inductive compound, named as OIC-A006 (Osteogenic inducible compound-active 006), which is classified in the amine family. In this study, we aimed to investigate the inducing effects of OIC-A006 on osteogenesis by bone marrow stem cells (BMSCs) in vitro and in vivo. We demonstrated that OIC-A006, at different concentrations, especially at optimal concentration of 6.25 microM, could stimulate BMSCs to express alkaline phosphatase (ALP), core-binding factor a1 (Cbfa1), osteopontin (OPN) and osteocalcin (OC), and to form calcified nodules in vitro. Under the bone tissue culture conditions, OIC-A006 also stimulated new bone formation of murine calvarial and metatarsal bone, indicating that OIC-A006 may exert positive effects on osteogenesis. Furthermore, to elucidate the in vivo osteogenic potential of OIC-A006, we used a rabbit skull defect model treated with sustained release microcapsules (OIC-A006/PLGA-MC) injected s.c. adjacent to the defect. These results revealed, for the first time, that OIC-A006 has the potential to promote osteogenesis in vitro and in vivo. This new compound may provide a new alterative agent for growth factors to promote bone healing and bone regeneration.