Shuo Geng

The Tumor Suppressor Role of miR-124 in Osteosarcoma

MicroRNAs have crucial roles in development and progression of human cancers, including osteosarcoma. Recent studies have shown that miR-124 was down-regulated in many cancers; however, the role of miR-124 in osteosarcoma development is unkonwn. In this study, we demonstrate that expression of miR-124 is significantly downregulated in osteosarcoma tissues and cell lines, compared to the adjacent tissues. The expression of miR-124 in the metastases osteosarcoma tissues was lower than that in non- metastases tissues. We identified and confirmed Rac1 as a novel, direct target of miR-124 using prdiction algorithms and luciferase reporter gene assays. Overexpression of miR-124 suppressed Rac1 protein expression and attenuated cell proliferation, migration, and invasion and induced apotosis in MG-63 and U2OS in vitro. Moreover, overexpression of Rac1 in miR-124-transfected osteosarcoma cells effectively rescued the inhibition of cell invasion caused by miR-124. Therefore, our results demonstrate that miR-124 is a tumor suppressor miRNA and suggest that this miRNA could be a potential target for the treatment of osteosarcoma in future.

miR-126 functions as a tumor suppressor in osteosarcoma by targeting Sox2.

Osteosarcoma (OS) is the most common malignant bone tumor in children and young adults, the early symptoms and signs of which are non-specific. The discovery of microRNAs (miRNAs) provides a new avenue for the early diagnosis and treatment of OS. miR-126 has been reported to be highly expressed in vascularized tissues, and is recently widely studied in cancers. Herein, we explored the expression and significance of miR-126 in OS. Using TaqMan RT-PCR analysis, we analyzed the expression of miR-126 in 32 paired OS tumor tissues and 4 OS cell lines and found that miR-126 was consistently under-expressed in OS tissues and cell lines compared with normal bone tissues and normal osteoblast cells (NHOst), respectively. As miR-126 is significantly decreased in OS tissues and cell lines, we sought to compensate for its loss through exogenous transfection into MG-63 cells with a miR-126 mimic. Ectopic expression of miR-126 inhibited cell proliferation, migration and invasion, and induced apoptosis of MG-63 cells. Moreover, bioinformatic prediction suggested that the sex-determining region Y-box 2 (Sox2) is a target gene of miR-126. Using mRNA and protein expression analysis, luciferase assays and rescue assays, we demonstrate that restored expression of Sox2 dampened miR-126-mediated suppression of tumor progression, which suggests the important role of miR-126/Sox2 interaction in tumor progression. Taken together, our data indicate that miR-126 functions as a tumor suppressor in OS, which exerts its activity by suppressing the expression of Sox2.

MicroRNA-224 promotes the sensitivity of osteosarcoma cells to cisplatin by targeting Rac1.

Osteosarcoma is the most common primary bone tumour in children and adolescents. Accumulating evidence has shown that microRNAs (miRNAs) participate in the development of almost all types of cancer. Here, we investigated the role of miR‐224 in the development and progression of osteosarcoma. We demonstrated that miR‐224 was down‐regulated in osteosarcoma cell lines and tissues. Lower miR‐224 levels were correlated with shorter survivalin osteosarcoma patients. Furthermore, overexpression of miR‐224 suppressed osteosarcoma cell proliferation, migration and invasion and contributed to the increased sensitivity of MG‐63 cells to cisplatin. We identified Rac1 as a direct target gene of miR‐224 in osteosarcoma. Rac1 expression was up‐regulated in the osteosarcoma cell lines and tissues, and there was an inverse correlation between Rac1 and miR‐224 expression in osteosarcoma tissues. Furthermore, rescuing Rac1 expression decreased the sensitivity of miR‐224‐overexpressing MG‐63 cells to cisplatin. We also demonstrated that ectopic expression of Rac1 promoted the proliferation, migration and invasion of miR‐224‐overexpressing MG‐63 cells. These data suggest that miR‐224 plays a tumour suppressor role in the development of osteosarcoma and is related to the sensitivity of osteosarcoma to cisplatin.

Modified distally based sural adipofascial flap for reconstructing of leg and ankle

While free flaps can be used in many cases to cover soft tissue defects in the distal leg and ankle in a single stage, factors such as diabetes and advanced age can interfere with success of vascular anastomoses.

A New Perspective for Osteosarcoma Therapy: Proteasome Inhibition by MLN9708/2238 Successfully Induces Apoptosis and Cell Cycle Arrest and Attenuates the Invasion Ability of Osteosarcoma Cells in Vitro

Background: The proteasome exists in all eukaryotic cells and provides the main route of intracellular proteins degradation involved in cell growth and apoptosis. Proteasome inhibition could block protein degradation pathways and disturb regulatory networks, possibly leading to profound effects on cell growth, particularly in cancer cells. A proteasome inhibitor with an appropriate toxicity index for malignant cells rather than normal cells would be an attractive anticancer therapy. Methods: The human osteosarcoma (OS) cell lines MG-63 and Saos-2 and normal osteoblast cells were used to study the antitumour activity of the proteasome inhibitor MLN9708/2238. Results: MLN2238 inhibited cell growth, induced cell cycle arrest and apoptosis, and attenuated the invasion abilities of MG-63 and Saos-2 cells, with little cytotoxicity to normal cells. In addition, MLN2238 promoted antitumour mechanisms including the accumulation of E2F1, P53, P21 and other negative G2/M checkpoint proteins; up-regulated the relative expression ratio of BAX/BCL-2, APAF-1 and pro-apoptotic proteins of the BCL-2 family; triggered mitochondrial outer membrane permeabilization (MOMP); down-regulated BCL-2 and XIAP; activated caspase3/8/9; and suppressed MMP2/9 expression and secretion levels. Conclusions: The proteasome may be a novel biochemical target for OS treatment in vitro. Our study provides a promising mechanistic framework for MLN9708/2238 in OS treatment, supporting its clinical development.

Steroid receptor co-activator-3 promotes osteosarcoma progression through up-regulation of FoxM1

Increasing evidence suggests that the three homologous members of steroid receptor co-activator (SRC) family (SRC-1, SRC-2, and SRC-3) play key roles in enhancing cell proliferation in various human cancers, such as breast, prostate, and hepatocellular carcinoma. However, the function of SRC-3 in osteosarcoma remains largely unexplored. In the current study, we found that SRC-3, but not SRC-1 and SRC-2, was dramatically up-regulated in human osteosarcoma tissues, compared with adjacent normal tissues. To explore the functions of SRC-3 in osteosarcoma, in vitro studies were performed in MG63 and U2OS cells. SRC-3 overexpression promoted osteosarcoma cell proliferation, whereas knockdown of SRC-3 inhibits its proliferation. In support of these findings, we further demonstrated that SRC-3 up-regulated FoxM1 expression through co-activation of C/EBPγ. Together our results show that SRC-3 drives osteosarcoma progression and imply it as a therapeutic target to abrogate osteosarcoma.

A Minimally Invasive Modified Reverse Sural Adipofascial Flap for Treating Posttraumatic Distal Tibial and Calcaneal Osteomyelitis

Our aim was to report a modified reverse sural adipofascial flap for treating posttraumatic distal tibial or calcaneal osteomyelitis. We retrospectively reviewed the records of 15 patients with posttraumatic distal tibial or calcaneal osteomyelitis treated with modified reverse sural adipofascial flaps between 2005 and 2010. The flap was raised through 2 short incisions in the posterior aspect of the lower leg. The raw surface of the flap was covered with a full-thickness skin graft. Donor sites were closed primarily. Lower Extremity Functional Scale (LEFS) scores and 2-point discrimination (TPD) were recorded preoperatively and postoperatively. There were 12 males and 3 females, with an average age of 39 years (range = 18-55 years). Twelve lesions were in the distal tibia and 3 in the calcaneus. The flap ranged in size from 11 × 5 cm to 16 × 7 cm. All flaps survived, and skin grafts healed without complications. Recipient sites had an anatomic contour, and all patients were able to ambulate without the assistance of special shoes or orthoses. No infections recurred, and no ulcers of the grafted skin occurred with the regular wearing of shoes. The follow-up duration was 18.7 ± 6.8 months (range = 12-36 months). The mean LEFS score increased from 22.4 ± 8.3 preoperatively to 53.0 ± 11.2 postoperatively (P = .001). TPD markedly recovered at 24 months postoperatively. The modified reverse sural adipofascial flap provides good outcomes in treating distal tibial and calcaneal osteomyelitis with minimal donor site morbidity.

Tetrahydrocurcumin induces mesenchymal-epithelial transition and suppresses angiogenesis by targeting HIF-1α and autophagy in human osteosarcoma

Human osteosarcoma is considered a malignant tumor with poor prognosis that readily metastasizes. Tetrahydrocurcumin (THC) has been reported to have anti-tumor activity in numerous tumors. In addition, hypoxia-inducible factor-1α (HIF-1α) has been demonstrated to be associated with tumor metastasis by regulating epithelial-mesenchymal transition (EMT). However, the role of THC in osteosarcoma remains uncertain. Therefore, this study aimed to elucidate the potential mechanisms. We found that THC significantly reduced the growth of osteosarcoma cells and suppressed migration and invasion, as tested in a nude mouse lung metastasis model. Additionally, the mesenchymal-epithelial transition (MET) process was facilitated by THC. Mechanistically, our study showed that HIF-1α had a pivotal role in the anti-metastatic effect of THC. Importantly, HIF-1α expression was downregulated by THC by inhibiting Akt/mTOR and p38 MAPK pathways. Moreover, THC exhibited a remarkable inhibitory effect on HIF-1α expression and angiogenesis under hypoxic conditions. Furthermore, THC activated autophagy and induced MET and suppressed angiogenesis in a HIF-1α-related manner. Taken together, our findings suggest that THC suppresses metastasis and invasion and this may be associated with HIF-1α and autophagy, which would potentially provide therapeutic strategies for human osteosarcoma.

Deoxyelephantopin Induces Reactive Oxygen Species-Mediated Apoptosis and Autophagy in Human Osteosarcoma Cells

Background/Aims: Osteosarcoma is the predominant form of primary bone malignancy. Although the combinational application of neoadjuvant chemotherapy and surgical resection significantly increases the survival rate, the therapeutic outcome remains unsatisfactory. Deoxyelephantopin (DET), an active ingredient of Elephantopus scaber, has been reported to have an anti-tumor effect in recent publications. This study aimed to investigate whether DET has antineoplastic effects on osteosarcoma cells and its underlying mechanism. Methods: Cell viability and morphological changes were assessed by MTT and Live/dead assays. Cell apoptosis, reactive oxygen species (ROS) and mitochondrial membrane potential were detected utilizing Annexin V-FITC/PI double staining, DCFH-DA and JC-1 probes, respectively. Autophagy was detected by mRFP-GFP-LC3 adenovirus transfection and western blot. Results: DET dose-dependently reduced the viability of osteosarcoma cells following the increase in intracellular ROS levels. Pretreatment with N-acetylcysteine (NAC) reversed this effect. Furthermore, DET induced mitochondrial apoptosis. Depolarized cells were increased, and apoptosis-related proteins, such as Bax, Bcl-2, cleaved caspase-9, cleaved caspase-3 and cleaved ploy ADP-ribose polymerase, were activated. Additionally, we found that DET could induce autophagy in osteosarcoma cells, but autophagy inhibition did not affect the decrease in cell viability. Conclusion: DET induced apoptosis in osteosarcoma cells through ROS generation, mitochondrial dysfunction and caspase activation; in addition, autophagy was involved in the effects of DET on osteosarcoma cells.

Apoptosis induced by sonodynamic therapy in human osteosarcoma cells in vitro

The aim of the present study was to investigate the potential effect of hematoporphyrin monomethyl ether-sonodynamic therapy (HMME-SDT) on MG-63 osteosarcoma cells. The HMME concentration was kept constant at 20 µg/ml and the MG-63 osteosarcoma cell line was exposed to ultrasound with an intensity of 1.0 W/cm2 for 30 sec. Cell cytotoxicity was quantified using an MTT assay 6 h after HMME-SDT. The intracellular localization of HMME was imaged using inverted confocal laser scanning microscopy. Apoptosis was investigated using flow cytometry with Annexin V-fluorescein isothiocyanate and propidium iodine staining. The cytotoxicity of HMME-mediated sonodynamic action on MG-63 cells was significantly higher than that of other treatments, including ultrasound alone, HMME alone and sham treatment. Flow cytometry demonstrated that HMME‑SDT action markedly enhanced the apoptotic rate of MG-63 cells. The mechanisms of apoptosis were analyzed by measuring the protein expression of poly ADP-ribose polymerase (PARP), cleaved PARP, procaspase-3, cleaved caspase-3 and cleaved caspase-9. The data demonstrated that HMME-SDT action markedly induced the apoptosis of MG-63 cells.