CircORC2 promoted proliferation and inhibited the sensitivity of osteosarcoma cell lines to cisplatin by regulating the miR-485-3p/TRIM2 axis.

Resistance to chemotherapy leads to poor prognosis for osteosarcoma (OS) patients. However, due to the high metastasis of tumor and the decrease in sensitivity of tumor cells to cisplatin (DDP), the 5-year survival rate of OS patients is still unsatisfactory. This study explored a mechanism for improving the sensitivity of OS cells to DDP. A DDP-resistant OS cell model was established, and we have found that circORC2 and TRIM2 were upregulated in DDP-resistant OS cells, but miR-485-3p was downregulated. The cell viability and proliferation of the OS cells decreased gradually with the increase of DDP dose, but a gradual increase in apoptosis was noted. CircORC2 promoted OS cell proliferation and DDP resistance and upregulated TRIM2 expression by targeting miR-485-3p. Functionally, circORC2 downregulated miR-485-3p to promote OS cell proliferation and inhibit DDP sensitivity. Additionally, it promoted cell proliferation and inhibited the sensitivity of DDP by regulating the miR-485-3p/TRIM2 axis. In conclusion, circORC2 promoted cell proliferation and inhibited the DDP sensitivity in OS cells via the miR-485-3p/TRIM2 axis. These findings indicated the role of circORC2 in regulating the sensitivity of OS cells to DDP.

Candidate Marker Genes for Diagnosis of Osteoarthritis and Prediction of Their Regulatory Mechanisms.

We have screened candidate marker genes for the diagnosis of osteoarthritis and predicted their regulatory mechanisms. Six expression chips of tissue samples and one expression chip of peripheral blood mononuclear cell (PMBC) samples were obtained from the GEO database. Differential analysis, GSEA, and WGCNA were performed on the integra-ted tissue sample data with batch correction. Can-didate genes were obtained from the intersection of the genes significantly related to osteoarthritis in the WGCNA and the differentially expressed genes. ROC analysis was performed on the candidate genes in the tissue and PMBC samples. Genes with AUC values greater than 0.6 were retained as final candidates, and their upstream regulatory miRNAs were predicted. A total of 106 genes with differential expression were found in osteoarthritis tissue samples, which were mainly enriched in cell cycle and p53 signalling pathways. WGCNA selected a gene module significantly correlated with the occurrence of osteoarthritis. Fourteen candidate genes were obtained from the intersection of the genes in the module and the differentially expressed genes. ROC analysis showed that among these 14 candidate genes, only ADM, CX3CR1 and GADD45A had AUC values greater than 0.6 in both tissue and PMBC samples. The AUC values of the gene set of these three genes were greater than 0.7. Multiple miRNAs were predicted to be regulators of these three genes. ADM, CX3CR1 and GADD45A have potential as diagnostic marker genes for osteoarthritis and may be regulated by multiple miRNAs.

Paeonol accelerates skin wound healing by regulating macrophage polarization and inflammation in diabetic rats.

Diabetic ulcer is usually seen in people with uncontrolled blood sugar. Reportedly, many factors such as impaired glucose metabolism, and macrovascular and microvascular diseases caused angiogenesis disorders and delayed the healing of diabetic ulcers, thus affecting the body's metabolism, nutrition, and immune function. This study aimed to explore the effect of paeonol on skin wound healing in diabetic rats and the related mechanism. A rat model of diabetic ulcer was established. High glucose-treated mouse skin fibroblasts were co-cultured with M1 or M2-polarized macrophages treated with or without paeonol. H&E and Masson staining were used to reveal inflammatory cell infiltration and collagen deposition, respectively. Immunohistochemistry visualized the expression of Ki67, CD31, and vascular endothelial growth factor (VEGF). Western blot was used to detect interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, IL-4, IL-10, CD31, VEGFA, and collagen I/III. The expression of iNOS and arginase 1 was revealed by immunofluorescence staining. Paeonol treatment augmented collagen deposition and the expression of Ki67, CD31, VEGF, and macrophage M2 polarization markers (IL-4 and IL-10) and reduced wound area, inflammatory cell infiltration, and macrophage M1 polarization markers (IL-1ß and TNF-α) in the ulcerated area. In vitro, paeonol treatment promoted M2-polarization and repressed M1-polarization in macrophages, thereby improving the repair of cell damage induced by high glucose. Paeonol accelerates the healing of diabetic ulcers by promoting M2 macrophage polarization and inhibiting M1 macrophage polarization.

EGCG Restores Keratinocyte Autophagy to Promote Diabetic Wound Healing through the AMPK/ULK1 Pathway.

BACKGROUND: Delayed wound healing, a common problem in patients with diabetes mellitus (DM), is associated with impaired keratinocyte autophagy. Epigallocatechin gallate (EGCG), a catechin, has been proven to promote diabetic wound healing. This study aims to explore the therapeutic mechanism of EGCG on diabetic wound healing. METHODS: High glucose (HG)-induced keratinocytes and streptozotocin (STZ)-induced DM rats were prepared and intervened with EGCG to examine its therapeutic effects in in vivo and in vitro settings. The AMPK inhibitor, Compound C, was utilized to determine whether EGCG exerted its therapeutic effects through the AMPK/ULK1 pathway. RESULTS: In vitro, EGCG improved HG-induced autophagy impairment in keratinocytes by increasing LC3II/LC3I, Becline1, and ATG5 levels and decreasing p62 level. Mechanically, EGCG activated the AMPK/ULK1 pathway, thereby promoting keratinocyte autophagy through the phosphorylation of AMPK and ULK1. Notably, EGCG promoted the proliferation, migration, synthesis and release of C-C motif chemokine ligand 2 (CCL2) in HG-treated keratinocytes. Furthermore, EGCG indirectly promoted the activation of fibroblasts, as evidenced by increased alpha-smooth muscle actin (α-SMA) and Collagen I levels. In vivo, EGCG promoted wound healing in DM rats, primarily by reducing inflammatory infiltration and increasing granulation tissue to promote wound epithelialization. Besides, EGCG promoted ATG5, KRT10, KRT14, TGF-ß1, Collagen I, and α-SMA expressions in the neonatal epithelial tissues of DM rats. However, the use of Compound C reversed the effects of EGCG. CONCLUSIONS: These findings indicated that EGCG restored keratinocyte autophagy to promote diabetic wound healing through the AMPK/ULK1 pathway.

miR-485-3p regulated by MALAT1 inhibits osteosarcoma glycolysis and metastasis by directly suppressing c-MET and AKT3/mTOR signalling.

AIMS: Osteosarcoma (OS) is an extremely malignant bone cancer with high incidence and rapid progression. This study aims to investigate the role and underlying mechanisms of MALAT1 and miR-485-3p in OS. MATERIALS AND METHODS: qRT-PCR and Western blotting were utilized to measure the levels of miR-485-3p, MALAT1, c-MET, AKT3, p-mTOR, mTOR, glycolysis-related proteins or migration-related proteins. Colony formation and transwell assay were used to test the roles of miR-485-3p, MALAT1, c-MET and AKT3 in cancer cell proliferation, migration and invasion. Dual luciferase assay was used to validate the interactions of miR-485-3p/c-MET, miR-485-3p/AKT3, and MALAT1/miR-485-3p. Glucose uptake assay and measurement of lactate production were employed to determine the glycolysis process. Mouse tumour xenograft model was used to determine the effect of shMALAT1 and miR-485-3p mimics on tumour growth and metastasis in vivo. KEY FINDINGS: miR-485-3p was decreased while c-MET, AKT3, and MALAT1 were increased in human OS tissues and cells. miR-485-3p bound directly to c-MET and AKT3 mRNAs and repressed OS cell glycolysis, proliferation, migration, and invasion through decreasing glycolysis-related proteins and migration-related proteins via inhibiting c-MET and AKT3/mTOR pathway. In addition, MALAT1 interacted with miR-485-3p and disinhibited c-MET and AKT3/mTOR signalling. Knockdown MALAT1 or overexpression of miR-485-3p restrained OS tumour growth and lung metastasis in vivo. SIGNIFICANCE: miR-485-3p suppresses OS glycolysis, proliferation, and metastasis via inhibiting c-MET and AKT3/mTOR signalling and MALAT1 acts as a sponge of miR-485-3p. MALAT1 and miR-485-3p may be the key regulators in OS progression, and potential molecular targets for future OS therapy.

Annular ligament reconstruction by suture anchor for treatment of radial head dislocation in children.

BACKGROUND: We investigated the efficacy of annular ligament reconstruction by suture anchor in the treatment of radial head dislocation (RHD) in children. METHOD: A total of 20 RHD children nderwent annular ligament reconstruction surgery using suture anchor. Preoperative and postoperative elbow functions were evaluated according to Broberg and Morrey 100-point scale. Recovery of radial nerve function was assessed using the Chinese Medical Association of Hand Surgery Branch of Upper Limb Functional Assessment Standard. All statistical analyses were performed using SPSS version 17.0 software. RESULTS: All 20 RHD children who underwent the procedure were followed up for a median duration of 24 months. At the last follow-up, the average Broberg-Morrey score was 94.3, with 12 children (60.0%) showing excellent outcomes (score range, 95 to 100), 7 children (35.0%) showing good outcomes (score range, 80 to 94), 1 child (5.0%) displayed a fair outcome (score range, 60 to 79), and 0 (0%) poor outcome. A significant difference in the excellent-good rate was observed when the elbow function before surgery was compared to after surgery (χ(2) = 5.559, P = 0.018). The radial nerve function of the 13 RHD children with radial nerve injury also recovered to normal. Among these 13 RHD children, nine exhibited excellent outcomes, 3 showed good outcomes, 1 displayed a fair outcome, and no patient showed a poor outcome. A significant difference in the excellent-good rate of radial nerve function was also observed when before surgery was compared to after surgery in these RHD children (χ(2) = 4.887, P = 0.027). CONCLUSION: Our results strongly indicated that suture anchor is highly effective for reconstruction of the annular ligament and to promote full functional recovery in RHD children, demonstrating that the procedure is an excellent treatment choice in RHD children.

MicroRNA-126 enhances the sensitivity of osteosarcoma cells to cisplatin and methotrexate.

The establishment of novel chemotherapy drugs for osteosarcoma is urgently required, and the mechanisms and effects of cisplatin (DDP) and methotrexate (MTX) in the current treatment of osteosarcoma have not been fully elucidated. The present study aimed to observe the effect of DDP, MTX and rapamycin on osteosarcoma cell proliferation and apoptosis, and to investigate the association between miR-126 and the effects of DDP and MTX in osteosarcoma cells. miR-126-overexpressing and -silencing lentiviral vectors were constructed, and MG63 and U-2 OS osteosarcoma cells were infected. An MTT assay was conducted to detect transfected cell proliferation, and the effects of the chemotherapy drugs on transfected cell apoptosis were detected by flow cytometry. The cell cycle of the transfected cells was analyzed via flow cytometry. As the miR-126-overexpressing and -silencing osteosarcoma cell lines were successfully constructed, it was observed that DDP and MTX inhibited osteosarcoma cell proliferation. With the decreased expression of miR-126, the sensitivity of osteosarcoma cells to DDP and MTX was reduced at the same concentration. The flow cytometry suggested that DDP and MTX could promote the apoptosis of osteosarcoma cells with overexpressed miR-126, whereas they could not significantly impact the apoptosis of the miR-126-silenced osteosarcoma cells. Meanwhile, DDP inhibited the cell cycle of the miR-126-overexpressing osteosarcoma cells. In conclusion, DDP and MTX inhibited the proliferation and promoted the apoptosis of the osteosarcoma cells, and these processes were dependent upon the expression of miR-126.

Overexpression of miR-126 sensitizes osteosarcoma cells to apoptosis induced by epigallocatechin-3-gallate.

BACKGROUND: miR-126 plays an important role in the proliferation, invasion, migration, and chemotherapeutics resistance in cancer. Epigallocatechin-3-gallate (EGCG), as the major polyphenolic constituent present in green tea, is a promising anticancer agent. However, the role of miR-126 in EGCG anticancer remains unclear. Here, we investigated the effects of miR-126 and EGCG on cell viability, apoptosis, cell cycle distribution of osteosarcoma cells and the sensitization of miR-126 on osteosarcoma cells to EGCG. METHODS: The cell viability, apoptosis and cycle distribution were analyzed using MTT assay and flow cytometry. RESULTS: Our results showed that EGCG (0.025, 0.05, 0.1, 0.2 g/L) suppresses proliferation of osteosarcoma MG63 and U2OS cells in a concentration-dependent and time-dependent manner and the inhibitory effects of 0.05 g/L EGCG on U2OS cells were roughly equivalent to 20 µM cisplatin (DDP); miR-126 could promote apoptosis and inhibit proliferation in U2OS cells but without significant effects on cell cycle G1 phase arrest; EGCG suppressed proliferation of U2OS cells through induction of cell cycle G1 arrest and apoptotic death; overexpression of miR-126 enhanced the inhibitory effects of EGCG on proliferation in U2OS cells via promotion of apoptosis. CONCLUSIONS: Our results demonstrate that enhanced expression of miR-126 increased the sensitivity of osteosarcoma cells to EGCG through induction of apoptosis.

miR-126 inhibits cell growth, invasion, and migration of osteosarcoma cells by downregulating ADAM-9.

Osteosarcoma (OS) has become one of the most common primary malignant tumors in the children and adolescents with a poor prognosis mainly due to high metastasis. A disintegrin and metalloprotease 9 (ADAM-9) plays a role in tumorigenesis, invasion, and metastasis in several tumors. miR-126 has been reported to be downregulated in OS tumor. However, the involvement of ADAM-9 in the pathology of OS and the relationship between miR-126 and ADAM-9 in OS cells remain unclear. In this study, using quantitative reverse-transcribed PCR (qRT-PCR) analysis on 37 pairs of OS tumors and matched adjacent normal bone tissues, we found that ADAM-9 is significantly upregulated, while miR-126 is downregulated in human OS tumors. Association analysis revealed that upregulation of ADAM-9 and downregulation of miR-126 are significantly involved in advanced clinical stage development and distant metastasis. Luciferase reporter assay revealed that miR-126 could directly target ADAM-9 3' untranslated region (UTR) and inhibit its expression in U2OS and MG-63 cells. Functional experiments revealed that downregulating ADAM-9 by miR-126 inhibited cellular growth, invasion, and migration in U2OS and MG-63 cells. In rescue experiments, restored ADAM-9 expression attenuated miR-126-mediated suppression, while knockdown of ADAM-9 by small interfering RNA (siRNA) represented similar results with miR-126-mediated tumor suppression in U2OS cells. Taken together, our data indicated that miR-126 inhibits cell growth, invasion, and migration of OS cells by downregulating ADAM-9.

Prognostic significance of p53 expression in malignant bone tumors: a meta-analysis.

Osteosarcoma and Ewing's sarcoma are the two most common primary malignant bone tumors, and findings of prognostic factors are important for clinicians to decide treatment options. High p53 expression has been implicated in tumor development and progression, but studies investigating the prognostic role of p53 overexpression in malignant bone tumors report conflicting findings. We performed a meta-analysis to assess the relationship between p53 overexpression and the survival of malignant bone tumors. A meta-analysis of 13 studies with a total of 703 patients was carried out to evaluate the association between p53 overexpression and overall survival (OS) and disease-free survival (DFS) in patients with malignant bone tumors. The pooled hazard ratio (HR) with its 95 % confidence interval (CI) was used as the effect size estimate. There was no between-study heterogeneity in both OS studies (I (2) = 0.0 %) and DFS studies (I(2) = 0.0 %). Overall, high p53 expression predicted both poor OS (HR 2.13, 95 % CI 1.81-2.52, P < 0.001) and poor DFS (HR 2.06, 95 % CI 1.58-2.69, P < 0.001) in patients with malignant bone tumors. Subgroup analyses by tumor types suggested that p53 expression predicted poor OS in both osteosarcoma patients (HR 2.15, 95 % CI 1.78-2.60, I (2) = 15.2 %, P < 0.001) and Ewing's sarcoma patients (HR 2.09, 95 % CI 1.47-2.97, I(2) = 0.0 %, P < 0.001). Besides, p53 expression also predicted poor DFS in both osteosarcoma patients (HR 2.38, 95 % CI 1.60-3.52, I(2) = 0.0 %, P < 0.001) and Ewing's sarcoma patients (HR 1.83, 95 % CI 1.28-2.63, I(2) = 0.0 %, P = 0.001). Egger's test also did not suggest evidence for publication bias in both OS studies (P = 0.615) and DFS studies (P = 0.258). High p53 expression indicates a poorer prognosis for patients with osteosarcoma and Ewing's sarcoma.