Mesenchymal Stem Cells-Involved Strategies for Rheumatoid Arthritis Therapy.

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic inflammation of the joints and bone destruction. Because of systemic administration and poor targeting, traditional anti-rheumatic drugs have unsatisfactory treatment efficacy and strong side effects, including myelosuppression, liver or kidney function damage, and malignant tumors. Consequently, mesenchymal stem cells (MSCs)-involved therapy is proposed for RA therapy as a benefit of their immunosuppressive and tissue-repairing effects. This review summarizes the progress of MSCs-involved RA therapy through suppressing inflammation and promoting tissue regeneration and predicts their potential clinical application.

Multifunctional hydrogel modulates the immune microenvironment to improve allogeneic spinal cord tissue survival for complete spinal cord injury repair.

Transplantation of allogeneic adult spinal cord tissues (aSCTs) to replace the injured spinal cord, serves as a promising strategy in complete spinal cord injury (SCI) repair. However, in addition to allograft immune rejection, damage-associated molecular pattern (DAMP)-mediated inflammatory microenvironments greatly impair the survival and function of transplants. In this study, we aimed to regulate the immune microenvironment after aSCT implantation by developing a functional hybrid gelatin and hyaluronic acid hydrogel (F-G/H) modified with cationic polymers and anti-inflammatory cytokines that can gelatinize at both ends of the aSCT to glue the grafts for perfect matching at defects. The F-G/H hydrogel exhibited the capacities of DAMP scavenging, sustainably released anti-inflammatory cytokines, and reduced lymphocyte accumulation, thereby modulating the immune response and enhancing the survival and function of aSCTs. When the hydrogel was used in combination with a systemic immunosuppressive drug treatment, the locomotor functions of SCI rats were significantly improved after aSCTs and F-G/H transplantation. This biomaterial-based immunomodulatory strategy may provide the potential for spinal cord graft replacement for treating SCI. STATEMENT OF SIGNIFICANCE: In this study, we aimed to regulate the immune microenvironment by developing a functional hybrid gelatin and hyaluronic acid hydrogel (F-G/H) modified with cationic polymers and anti-inflammatory cytokines that can gelatinize at both ends of the aSCT to glue the grafts for perfect matching at defects. We found that with the treatment of F-G/H hydrogel, the aSCT survival and function was significantly improved, as a result of reducing recruitment and activation of immune cells through TLR- and ST-2- related signaling. With the combination of immunosuppressive drug treatment, the locomotor functions of SCI rats were significantly improved after aSCTs and F-G/H transplantation. Findings from this work suggest the potential application of the F-G/H as a biomaterial-based immunoregulatory strategy for improving the therapeutic efficiency of the transplanted spinal cord graft for spinal cord injury repair.

2-BFI Provides Neuroprotection Against Fluorosis by Stabilizing Endoplasmic Reticulum-Mitochondria Contact Sites and Inhibiting Activation of the NLRP3 Inflammasome.

2-(2-benzofu-ranyl)-2-imidazoline (2-BFI) is a drug that has attracted much attention in recent years. It has a therapeutic effect on brain diseases in animal models such as Alzheimer's disease and cerebral infarction. However, whether 2-BFI affords neuroprotection against the toxicity of fluoride, which can cross the blood-brain barrier and cause neurological dysfunction is not known. We investigated the cell viability and apoptosis of SH-SY5Y cells and primary cultures of cortical neurons exposed to fluoride, and 2-BFI was used to protect both two kinds of cells against the effects of fluoride. We found that 2-BFI can provide neuroprotection on SH-SY5Y cells and primary cultures of cortical neurons upon fluorosis by maintaining the stability of endoplasmic reticulum-mitochondria contact sites and inhibiting activation of the NLR family pyrin domain containing 3 (NLRP3) inflammasome. This study may provide a new method for protecting against the neurotoxicity induced by fluoride exposure.

Acidity-Triggered Transformable Polypeptide Self-Assembly to Initiate Tumor-Specific Biomineralization.

Biomineralization is a normal physiological process that includes nucleation, crystal growth, phase transformation, and orientation evolution. Notably, artificially induced biomineralization in the tumor tissue has emerged as an unconventional yet promising modality for malignancy therapy. However, the modest ion-chelating capabilities of carboxyl-containing biomineralization initiators lead to a deficient blockade, thus compromising antitumor efficacy. Herein, a biomineralization-inducing nanoparticle (BINP) is developed for blockade therapy of osteosarcoma. BINP is composed of dodecylamine-poly((γ-dodecyl-l-glutamate)-co-(l-histidine))-block-poly(l-glutamate-graft-alendronate) and combines a cytomembrane-insertion moiety, a tumor-microenvironment (TME)-responsive component, and an ion-chelating motif. After intravenous injection into osteosarcoma-bearing mice, BINP responds to the acidic TME to expose the dodecyl group on the surface of the expanded nanoparticles, facilitating their cytomembrane insertion. Subsequently, the protruding bisphosphonic acid group triggers continuous ion deposition to construct a mineralized barrier around the tumor, which blocks substance exchange between the tumor and surrounding normal tissues. The BINP-mediated blockade therapy displays tumor inhibition rates of 59.3% and 52.1% for subcutaneous and orthotopic osteosarcomas, respectively, compared with the Control group. In addition, the suppression of osteoclasts by the alendronate moiety alleviates bone dissolution and further inhibits pulmonary metastases. Hence, the BINP-initiated selective biomineralization provides a promising alternative for clinical osteosarcoma therapy.

Osteoimmunity-Regulating Biomimetically Hierarchical Scaffold for Augmented Bone Regeneration.

Engineering a proper immune response following biomaterial implantation is essential to bone tissue regeneration. Herein, a biomimetically hierarchical scaffold composed of deferoxamine@poly(ε-caprolactone) nanoparticles (DFO@PCL NPs), manganese carbonyl (MnCO) nanosheets, gelatin methacryloyl hydrogel, and a polylactide/hydroxyapatite (HA) matrix is fabricated to augment bone repair by facilitating the balance of the immune system and bone metabolism. First, a 3D printed stiff scaffold with a well-organized gradient structure mimics the cortical and cancellous bone tissues; meanwhile, an inside infusion of a soft hydrogel further endows the scaffold with characteristics of the extracellular matrix. A Fenton-like reaction between MnCO and endogenous hydrogen peroxide generated at the implant-tissue site triggers continuous release of carbon monoxide and Mn2+ , thus significantly lessening inflammatory response by upregulating the M2 phenotype of macrophages, which also secretes vascular endothelial growth factor to induce vascular formation. Through activating the hypoxia-inducible factor-1α pathway, Mn2+ and DFO@PCL NP further promote angiogenesis. Moreover, DFO inhibits osteoclast differentiation and synergistically collaborates with the osteoinductive activity of HA. Based on amounts of data in vitro and in vivo, strong immunomodulatory, intensive angiogenic, weak osteoclastogenic, and superior osteogenic abilities of such an osteoimmunity-regulating scaffold present a profound effect on improving bone regeneration, which puts forward a worthy base and positive enlightenment for large-scale bone defect repair.

The effects of cages implantation on surgical and adjacent segmental intervertebral foramina.

OBJECTION: The overarching goal of our research was to compare the clinical and radiological outcomes with different sizes of cages implantation in anterior cervical discectomy and fusion (ACDF), and to evaluate the effects on surgical and adjacent segmental intervertebral foramina. METHODS: The clinical data of 61 patients were analyzed retrospectively. The radiological data included the surgical intervertebral disk space height before (H0) and after surgery (H), the preoperative mean height of adjacent segments (Hm), the area and height of the surgical and adjacent segment foramen, the surgical segmental Cobb angle (α1), and C2-7Cobb angle (α2). The calculation of clinical data was conducted by Japanese Orthopaedic Association Scores (JOA), the recovery rate of JOA scores and visual analog scales (VAS). In accordance with the different ranges of distraction (H/Hm), patients were classified into three groups: group A (H/Hm<1.20, n=13), group B (1.20≤H/Hm≤1.80, n=37), and group C (H/Hm>1.80, n=11). RESULTS: After the operation and at the final follow-up, our data has demonstrated that the area and height of surgical segmental foramen all increased by comparing those of preoperation in three groups (all P<0.05). However, except for a decrease in group C (all P<0.05), the adjacent segmental foramina showed no significant changes (all P>0.05). The area and height of the surgical segment foramen and the distraction degree were positively correlated (0

Down-Regulation of microRNA-30d Alleviates Intervertebral Disc Degeneration Through the Promotion of FOXO3 and Suppression of CXCL10.

MicroRNAs (miRNAs/miRs) are important biomarkers for the progression of intervertebral disc degeneration (IDD). We investigated the role of miR-30d in IDD progression through its interactions with forkhead box O3 (FOXO3) and C-X-C motif ligand 10 (CXCL10). We first measured the expression of miR-30d, FOXO3, and CXCL10 in NP cells cultured from IDD patients. RNA-immunoprecipitation (RIP), chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays were then employed to test the relationship among miR-30d, FOXO3, and CXCL10. Besides, gain- and loss-of function approaches were performed to assess the functional roles of miR-30d and FOXO3 in IDD in vitro and in vivo. We found high expression of miR-30d and CXCL10 and low expression of FOXO3 in IDD. We showed that miR-30d specifically targeted FOXO3, and that down-regulation of miR-30d promoted proliferation and inhibited apoptosis of NP cells in IDD by increasing the expression of FOXO3. Besides, FOXO3 inhibited apoptosis of NP cells by downregulation of CXCL10 expression. Moreover, inhibition of miR-30d promoted proliferation and inhibited apoptosis of NP cells in IDD by decreasing CXCL10. Furthermore, findings in the mouse IDD model confirmed the inhibitory role of decreased miR-30d in IDD progression. Thus, we show that downregulation of miR-30d could promote the proliferation of NP cells by increasing FOXO3 and decreasing CXCL10 expression, which may provide a novel therapeutic target for IDD.

Thermogel Delivers Oxaliplatin and Alendronate in situ for Synergistic Osteosarcoma Therapy.

The therapeutic effect of osteosarcoma (OS) has not made extraordinary progress in the past few decades. Oxaliplatin (OXA) is a widely used clinical anti-tumor drug. Recent studies have shown that OXA can trigger anti-tumor immunity by inducing immunogenic death (ICD). Alendronate (ALN) has been used to threaten the skeletal system tumors because of the unique bone affinity and the ability to inhibit bone destruction. In this study, we co-loaded OXA and ALN on mPEG45-PLV19 thermo-sensitive hydrogel to perform in situ treatment on the mouse OS model. Slowly released OXA can induce immunogenic death of tumor cells. At the same time, thermo-sensitive hydrogels can induce the accumulation of cytotoxic T lymphocytes. Besides, ALN could synergistically diminish tumors and prevent bone destruction. This system could synergistically inhibit the progression of OS and lung metastasis and has no toxicity to various organs throughout the body.

Construction of an integrated human osteosarcoma database, HOsDb, based on literature mining, microarray analysis, and database retrieval.

BACKGROUND: Osteosarcoma (OS) is the most frequent primary malignancy of bone with a high incidence in adolescence. This study aimed to construct a publicly available, integrated database of human OS, named HOsDb. METHODS: Microarray data, current databases, and a literature search of PubMed were used to extract information relevant to human OS-related genes and their transcription factors (TFs) and single nucleotide polymorphisms (SNPs), as well as methylation sites and microRNAs (miRNAs). This information was collated for constructing the HOsDb. RESULTS: In total, we identified 7191 OS tumor-related genes, 763 OS metastasis-related genes, and 1589 OS drug-related genes, corresponding to 190,362, 21,131, and 41,135 gene-TF pairs, respectively, 3,749,490, 358,361, and 767,674 gene-miRNA pairs, respectively; and 28,386, 2532, and 3943 SNPs, respectively. Additionally, 240 OS-related miRNAs, 1695 genes with copy number variations in OS, and 18 genes with methylation sites in OS were identified. These data were collated to construct the HOsDb, which is available at www.hosdatabase.com. Users can search OS-related molecules using this database. CONCLUSION: The HOsDb provides a platform that is comprehensive, quick, and easily accessible, and it will enrich our current knowledge of OS.

lncRNA CEBPA-AS1 Overexpression Inhibits Proliferation and Migration and Stimulates Apoptosis of OS Cells via Notch Signaling.

Osteosarcoma (OS) is the most common primary bone malignancy derived from primitive bone-forming mesenchymal cells. Long noncoding RNA (lncRNA) expression profiles have been intensively studied for their involvement in OS. Herein, we clarify whether lncRNA CEBPA-AS1 is a regulator of NCOR2 in OS cells. Microarray-based expression analysis identified OS-related differentially expressed lncRNA and predicted microRNAs (miRs) binding to lncRNA and mRNA. lncRNA CEBPA-AS1 and NCOR2 were found to be weakly expressed in OS tissues and cells. Next, functional investigation revealed that lncRNAs CEBPA-AS1 bound to miR-10b-5p to upregulate NCOR2. Following that, gene-targeted knockdown and overexpressed recombinant vectors of lncRNA CEBPA-AS1 and NCOR2 were constructed to explore the effects of lncRNA CEBPA-AS1 and NCOR2 on cell proliferation, differentiation, migration, and apoptosis. Finally, tumor formation was measured in nude mice. lncRNA CEBPA-AS1 overexpression or NCOR2 elevation inhibited cell proliferation and migration, and alkaline phosphatase (ALP) and bone gla protein (BGP) activity, while enhancing apoptosis and tumor formation. Furthermore, NCOR2 was elevated in response to lncRNA CEBPA-AS1 overexpression, thus repressing the Notch signaling pathway. Taken together, lncRNA CEBPA-AS1 overexpression inhibits OS progression through diminishing activation of the Notch signaling pathway via upregulating NCOR2. Therefore, lncRNA CEBPA-AS1 may serve as a molecular target for treating OS.

Down-regulation of RAC2 by small interfering RNA restrains the progression of osteosarcoma by suppressing the Wnt signaling pathway.

Osteosarcoma (OS) is the most common primary malignancy of bone and is characterized by a high malignant and metastatic potential. Microarray-based differentially expressed gene screening determined RAC2 as the candidate gene related to OS. Highly expressed RAC2 and activated Wnt signaling pathway were determined in OS tissues using reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis. The OS cells were transfected with siRNA-RAC2 or treated with BIO (activator of Wnt pathway), whereby the effects of siRNA-RAC2 on cell proliferation, invasion, cycle and apoptosis were analyzed by CCK-8, Transwell assay and flow cytometry. The mRNA and protein levels of RAC2 and the Wnt signaling pathway-, proliferation- and apoptosis-related genes in OS cells were determined by RT-qPCR and Western blot assay. Importantly, siRNA-mediated RAC2 silencing inhibited the activation of the Wnt signaling pathway in OS. siRNA-RAC2 inhibited the proliferation and invasion, while impeded OS cell cycle progression and facilitated cell apoptosis. However, activation of Wnt signaling pathway reversed the effects of siRNA-RAC2. Finally, orthotopic xenograft OS mouse model confirmed the in vivo anti-tumor effects by silencing RAC2. Taken together, RAC2 gene silencing could suppress OS progression. The mechanism was obtained by inhibiting the activation of the Wnt signaling pathway.

MicroRNA-200c promotes osteogenic differentiation of human bone mesenchymal stem cells through activating the AKT/ß-Catenin signaling pathway via downregulating Myd88.

During the human bone formation, the event of osteogenic differentiation of human bone mesenchymal stem cells (hBMSCs) is vital, and recent evidence has emphasized the important role of microRNAs (miRNAs) in osteogenic differentiation of hBMSCs. This study aims to examine the potential effects of miR-200c in osteogenic differentiation of hBMSCs and understand their underlying mechanisms. HBMSCs were obtained via human bone marrow. During osteogenic induction and differentiation, cells were transfected with different plasmids with the intention of investigating the roles of miR-200c on osteogenic differentiation, calcium salt deposition, alkaline-phosphatase (ALP) activity, mineralized nodule formation, osteocalcin (OCN) content, and proliferation of osteoblasts. Following transfection, dual luciferase reporter gene assay was conducted so as to explore the correlation between miR-200c and Myd88. Moreover, the AKT/ß-Catenin signaling pathway was blocked with an AKT/ß-Catenin inhibitor, AKTi, to investigate its involvement. The hBMSCs were successfully isolated from human bone marrow. Myd88 was determined as a target gene of miR-200c. Gain and loss-of-function assays confirmed that overexpression of miR-200c, or silencing of Myd88 promoted osteogenic differentiation, increased calcium salt deposition, ALP activity, mineralized nodule formation, and enhanced the proliferation of osteoblasts following osteogenic differentiation of hBMSCs. Meanwhile, the downregulation of miR-200c has been shown to have the opposite effect. Furthermore, these findings showed that the miR-200c overexpression activated the AKT/ß-Catenin signaling pathway by targeting Myd88. To sum up, the miR-200c upregulation induces osteogenic differentiation of hBMSCs by activating the AKT/ß-Catenin signaling pathway via the inhibition of Myd88, providing a target for treatment of bone repair.

MicroRNA-377 exerts a potent suppressive role in osteosarcoma through the involvement of the histone acetyltransferase 1-mediated Wnt axis.

It has been demonstrated that microRNAs (miRNAs) may contribute to tumorigenesis and tumor growth in osteosarcoma (OS), which is a primary malignant tumor of bone frequently diagnosed in adolescents and young people. The purpose of our investigation was to evaluate the functional relevance of miR-377 in OS and to investigate whether the mechanism was related to the histone acetyltransferase 1 (HAT1)-mediated Wnt signaling pathway. By screening differentially expressed genes in microarray GSE47572, HAT1 was found to be a candidate gene of interest. Besides, the regulatory miRNA (miR-377) of HAT1 was also selected. The interaction among miR-377, HAT1, and the Wnt signaling pathway was evaluated. In addition, the miR-377 expression was altered in OS cells (U-2OS and SOSP-9607) to assess the in vitro cell apoptosis and the in vivo tumor growth. OS tissues presented elevated HAT1 expression and decreased miR-377 expression. A putative miR-377 binding site in HAT1 3'-UTR HAT1 was verified. Cells with miR-377 overexpression or HAT1 silencing were observed to exhibit reduced HAT1 expression and promoted apoptosis, accompanied by blockade of Wnt signaling. Moreover, the in vivo experiment revealed that miR-377 overexpression or HAT1 silencing inhibited tumor growth and reduced tumor size in nude mice. Taken together, our results conclude that miR-377 may promote OS cell apoptosis through inactivation of the HAT1-mediated Wnt signaling pathway, highlighting the potential therapeutic effect of miR-377 on OS treatment.

Laden Nanofiber Capsules for Local Malignancy Chemotherapy.

In contrast to systemic chemotherapy, the local administration of chemotherapeutics potentially optimizes their accumulation in the tumor tissues and alleviates the associated systemic toxicity. However, the lack of control over the release of encapsulated drugs and their degradation represents a major challenge for local drug delivery systems. In this study, we developed a series of epirubicin (EPI)-loaded poly(ε-caprolactone)/poly(lactide-co-glycolide) (PCL/PLGA) nanofiber capsules with adjustable rates of drug release and degradation for local malignancy chemotherapy. The core-sheath PCL/PLGA nanofiber capsules containing 15.0 or 25.0 wt% of PCL in the sheath and 0, 5.0, or 10.0 wt% of EPI in the core were produced by emulsion electrospinning technology. The EPI release and degradation of the nanofiber capsules could be accelerated by decreasing the PCL content in the sheath. More importantly, the EPI-loaded PCL/PLGA nanofiber capsules effectively inhibited the growth of tumor cells in vitro and in vivo. Moreover, none of the laden nanofiber capsules caused apparent systemic toxicity. Hence, the cytostatic-loaded electrospun polyester nanofiber capsules displayed controlled drug release and degradation, along with satisfactory antitumor properties, indicating their great potential in local malignancy chemotherapy.

Effects of FOSL1 silencing on osteosarcoma cell proliferation, invasion and migration through the ERK/AP-1 signaling pathway.

Osteosarcoma (OS), as the most frequent primary malignancy of bone, is characterized by the presence of malignant mesenchymal cells. In the current study, our aim was to explore the possible effects Fos-like antigen-1 (FOSL1) had on the silencing regarding OS cell proliferation, invasion, and migration through the activation of the extracellular-signal-regulated kinase (ERK)/activator protein-1 (AP-1) signaling pathway. After the collection of OS on top of already having the adjacent normal tissue samples, the protein positive expression rate of FOSL1 was then measured by implementing the use of immunohistochemistry and discovered that FOSL1 was robustly expressed in OS. Later, to better grasp the impact FOSL1 projects on OS and its underlying mechanism, we determined the OS related genes as well as the ERK/AP-1 signaling pathway related genes expression by using a reverse-transcription quantitative polymerase chain reaction and western blot assay techniques. The results of the aforementioned two experiments revealed that the FOSL1 depletion had downregulated the expression of OS related genes by simultaneously downregulating the ERK/AP-1 signaling pathway. Moreover, cell proliferation, cycle, apoptosis, invasion, and migration of FOS1 were all tested by using a cell counting kit-8 assay, flow cytometry, Transwell assay, and scratch test, and these results presented that silencing of the FOSL1 gene inhibited OS cell proliferation, invasion, and migration. Our findings revealed a novel mechanism by which FOSL1 depletion played a significantly negative role in the OS progression through the regulation of the ERK/AP-1 signaling pathway. Functional suppression of FOSL1 might be a future therapeutic strategy regarding OS.

Does serum alkaline phosphatase level really indicate the prognosis in patients with osteosarcoma? A meta-analysis.

OBJECTIVE: Osteosarcoma is the most common malignant bone tumor that occurs in children and adolescents. Various studies have investigated the role of serum alkaline phosphatase (ALP) level in patients with osteosarcoma but report conflicting findings. Thus, a meta-analysis to assess its prognostic value more precisely is conducted. MATERIALS AND METHODS: Pooled hazard ratio (HR) with 95% confidence intervals (CIs) of overall survival (OS) was used to assess the prognostic role of serum ALP level. Eleven studies published between 1993 and 2013 with a total of 1336 osteosarcoma patients were included. RESULTS: Overall, the pooled HR for all 11 eligible studies evaluating high ALP level on OS was 1.60 (95% CI: 1.38-1.86). Sensitivity analysis suggested that the pooled HR was stable and omitting a single study did not change the significance of the pooled HR. Begg's (0.553) and Egger's (0.382) test also did not suggest evidence for publication bias. CONCLUSION: This meta-analysis suggests that high serum ALP level is obviously associated with lower OS rate in patients with osteosarcoma, and it is an effective biomarker of prognosis.

P53 is required for Doxorubicin-induced apoptosis via the TGF-beta signaling pathway in osteosarcoma-derived cells.

Osteosarcoma is the most common type of aggressive bone cancer. Current treatment strategies include surgical resection, radiation, and chemotherapy. Doxorubicin has been widely used as a chemotherapeutic drug to treat osteosarcoma. However, drug resistance has become a challenge to its use. In this study, p53-wild type U2OS and p53-null MG-63 osteosarcoma-derived cells were used to investigate the mechanism of doxorubicin-induced cytotoxicity. In cell viability assays, doxorubicin effectively induced apoptosis in U2OS cells via the p53 signaling pathway, evidenced by elevated PUMA and p21 protein levels and activated caspase 3 cleavage. In contrast, p53-null MG-63 cells were resistant to doxorubicin-induced apoptosis, while exogenous expression of p53 increased drug sensitivity in those cells. The role of TGF-ß/Smad3 signaling was investigated by using TGF-ß reporter luciferase assays. Doxorubicin was able to induce TGF-ß signal transduction without increasing TGF-ß production in the presence of p53. Knockdown of Smad3 expression by small hairpin RNA (shRNA) showed that Smad3 was required for p53-mediated TGF-ß signaling in response to doxorubicin treatment in U2OS and MG-63 cells. Taken together, these data demonstrate that p53 and TGF-ß/Smad3 signaling pathways are both essential for doxorubicin-induced cytotoxicity in osteosarcoma cells.

Biological roles of microRNA-140 in tumor growth, migration, and metastasis of osteosarcoma in vivo and in vitro.

The objective of this study was to explore the biological roles of microRNA-140 (miR-140) in tumor growth, migration, and metastasis of osteosarcoma (OS) in vivo and in vitro. Between 2007 and 2014, 47 cases of OS samples and normal bone tissue samples adjacent to OS were selected from our hospital. Tissue biopsies from OS patients were used to measure miR-140 levels to obtain a correlation between clinicopathological features and miR-140 expression. In vitro, MG63 human osteosarcoma cells were divided into four groups: blank group, miR-140 mimic group, miR-140 inhibitor group, and negative control (NC; empty plasmid) group. qRT-PCR was used to detect miR-140 expression, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to detect cell proliferation, flow cytometry was used to detect cell cycle distribution, and scratch migration assay was used to detect cell migration. In vivo, the relative expression of miR-140 level in OS tissue was lower than that in the adjacent normal bone tissue. miR-140 expression is inversely correlated with tumor size, Enneking stage, and tumor metastasis. In vitro, compared with blank group and NC group, relative miR-140 expression was increased, cell proliferation was inhibited, cell population in G0/G1 phase was increased, cell population in G2/M phase and S phases and proliferation index (PI), and cell migration distance were decreased in the miR-140 mimic group, but the relative expression and all the cell indexes were found opposite trend in the miR-140 inhibitor group. In conclusion, in vivo and vitro findings provided evidence that miR-140 could inhibit the growth, migration, and metastasis of OS cells.

p53 mediated apoptosis in osteosarcoma MG-63 cells by inhibition of FANCD2 gene expression.

PURPOSE: The aim of this study was to investigate the association between osteosarcoma (OS) and Fanconi anemia (FA) related pathways and the molecular mechanisms. METHODS: siRNA for Fanconi anemia complementation group D2 (FANCD2) was constructed and transfected into the osteosarcoma cell line MG-63 cells. Expression of TP53INP1, p53, p21, caspase-9, and caspase-3 mRNA in MG-63 cells were examined by real-time fluorescence quantitative PCR, and the protein levels were also determined by western blot. RESULTS: After silence of the FANCD2 gene in MG-63 cells, cell proliferation was inhibited, cell cycle was arrested and cell apoptosis was induced. The apoptosis was mediated by the p53 signaling pathway. After FANCD2 expression was inhibited, TP53INP1 gene expression was up-regulated, phosphorylation of p53 was promoted and the p21 protein was activated, leading to cell cycle arrested in G1, finally resulted in caspase-dependent cell apoptosis. CONCLUSIONS: Inhibition of FANCD2 gene expression can induce apoptosis of osteosarcoma cells, which indicated that FANCD2 played an important role in the development of osteosarcoma and it might be a potential target for treatment of osteosarcoma.