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.

HSP90 regulates osteosarcoma cell apoptosis by targeting the p53/TCF-1-mediated transcriptional network.

Osteosarcoma (OS) is the most common bone tumor that occurs predominantly in children and teenagers. Although many genes, such as p53 and Rb1, have been shown to be mutated, deregulation of the canonical Wnt/ß-catenin signaling pathway is frequently observed in OS. We recently demonstrated that heat shock protein 90 (HSP90) is involved in the regulation of runt-related transcription factor 2 via the AKT/GSK-3ß/ß-catenin signaling pathway in OS. However, the precise role of T cell factors/lymphoid enhancer-binding factor (TCFs/LEF) family members, which are the major binding complex of ß-catenin, in OS is poorly understood. In the present study, we first demonstrated that TCF-1 is overexpressed in OS compared with other bone tumors. Knockdown of TCF-1 significantly induced cell cycle arrest, severe DNA damage, and subsequent caspase-3-dependent apoptosis. Interestingly, coexpression of HSP90 and TCF-1 was observed in OS, and mechanistically, we demonstrated that TCF-1 expression is regulated by HSP90 either through a ß-catenin-dependent mechanism or a direct degradation of the proteasome. We also found that overexpression of TCF-1 partially abolishes the apoptosis induced by HSP90 inhibition. Furthermore, we provided evidence that p53, but not miR-34a, plays a crucial role in the HSP90-regulated TCF-1 expression and subsequent apoptosis. Given the diverse combination regimens of HSP90 inhibition with some other treatments, we propose that the p53 status and the expression level of TCF-1 should be taken into consideration to enhance the therapeutic efficacy of HSP90 inhibition.

Atomic Force Microscopy-Based Nanoscopy of Chondrogenically Differentiating Human Adipose-Derived Stem Cells: Nanostructure and Integrin ß1 Expression.

Integrin ß1 is known to be involved in differentiation, migration, proliferation, wound repair, tissue development, and organogenesis. In order to analyze the binding probability between integrin ß1 ligand and cluster of differentiation 29 (CD29) receptors, atomic force microscopy (AFM) was used to detect native integrin ß1-coupled receptors on the surface of human adipose-derived stem cells (hADSc). The binding probability of integrin ß1 ligand-receptor interaction was probed by integrin ß1-functionalized tips on hADSc during early chondrogenic differentiation at the two-dimensional cell culture level. Cell morphology and ultrastructure of hADSc were measured by AFM, which demonstrated that long spindled cells became polygonal cells with decreased length/width ratios and increased roughness during chondrogenic induction. The binding of integrin ß1 ligand and CD29 receptors was detected by ß1-functionalized tips for living hADSc. A total of 1200 curves were recorded at 0, 6, and 12 days of chondrogenic induction. Average rupture forces were, respectively, 61.8 ± 22.2 pN, 60 ± 20.2 pN, and 67.2 ± 22.0 pN. Rupture events were 19.58 ± 1.74%, 28.03 ± 2.05%, and 33.4 ± 1.89%, respectively, which demonstrated that binding probability was increased between integrin ß1 ligand and receptors on the surface of hADSc during chondrogenic induction. Integrin ß1 and the ß-catenin/SOX signaling pathway were correlated during chondrogenic differentiation. The results of this investigation imply that AFM offers kinetic and visual insight into the changes in integrin ß1 ligand-CD29 receptor binding on hADSc during chondrogenesis. Changes in cellular morphology, membrane ultrastructure, and the probability of ligand-transmembrane receptor binding were demonstrated to be useful markers for evaluation of the chondrogenic differentiation process.

Transcriptional regulation of Runx2 by HSP90 controls osteosarcoma apoptosis via the AKT/GSK-3ß/ß-catenin signaling.

Osteosarcoma (OS) is the most malignant primary bone tumor in children and adolescents with limited treatment options and poor prognosis. Recently, aberrant expression of Runx2 has been found in OS, thereby contributing to the development, and progression of OS. However, the upstream signaling molecules that regulate its expression in OS remain largely unknown. In the present study, we first confirmed that the inhibition of HSP90 with 17-AAG caused significant apoptosis of OS cells via a caspase-3-dependent mechanism, and that inhibition or knockdown of HSP90 by 17-AAG or siRNAs significantly suppressed mRNA and protein expression of Runx2. Furthermore, we provided evidence that Runx2 was transcriptionally regulated by HSP90 when using MG132 and CHX chase assay. We also demonstrated that ß-catenin was overexpressed in OS tissue, and that knockdown of ß-catenin induced pronounced apoptosis of OS cells in the presence or absence of 17-AAG. Interestingly, this phenomenon was accompanied with a significant reduction of Runx2 and Cyclin D1 expression, indicating an essential role of Runx2/Cyclin D1 in 17-AAG-induced cells apoptosis. Moreover, we demonstrated that the apoptosis of OS cells induced by 17-AAG did require the involvement of the AKT/GSK-3ß/ß-catenin signaling pathway by using pharmacological inhibitor GSK-3ß (LiCl) or siGSK-3ß. Our findings reveal a novel mechanism that Runx2 is transcriptionally regulated by HSP90 via the AKT/GSK-3ß/ß-catenin signaling pathway, and by which leads to apoptosis of OS cells.

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.