Migration Of MG-63 Cells Research Articles

MiR-18a-5p promotes cell invasion and migration of osteosarcoma by directly targeting IRF2.

An increasing number of studies have suggested that microRNAs (miRNAs) are involved in the progress of many human cancers including osteosarcoma (OS). Especially, microRNA-18a-5p (miR-18a-5p) has been reported to associate with the occurrence, development and clinical outcomes of human cancers. Therefore, we investigated the functions of miR-18a-5p in OS. Reverse transcription-quantitative PCR (RT-qPCR) showed that miR-18a-5p was significantly upregulated in OS tissues and cell lines (MG-63 and Saos-2). The overexpression of miR-18a-5p was found to significantly promote cell migration and invasion in MG-63 cells via Transwell assay. Moreover, luciferase reporter assays indicated that interferon regulatory factor (IRF)2 was a direct target of miR-18a-5p. IRF2 was downregulated in MG-63 and Saos-2 cell lines. Furthermore, Transwell analysis showed that the knockout of IRF2 promoted cell migration and invasion in MG-63 cells. Carcinogenesis of miR-18a-5p was reversed by the overexpression of IRF2 in OS. In conclusion, miR-18a-5p promoted the invasion and migration of OS cells through inhibiting IRF2 expression. Thus, miR-18a-5p might act as a potential target for the diagnosis and treatment of OS in the future.

Hypoxia-inducible factor-1 promotes cancer progression through activating AKT/Cyclin D1 signaling pathway in osteosarcoma.

Osteosarcoma is an aggressive malignant neoplasm, which commonly afflicts patients of 20-30 years of age, and its morbidity has increased markedly in recent years. Certain genes and signal pathways have been identified to exert key roles in osteosarcoma progression. Here, we set out to characterize in more detail of the role of HIF-1/AKT/Cyclin D1 pathway in the progression of osteosarcoma. Immunohistochemistry, western blot and qPCR were used to test the protein or mRNA levels of HIF-1 in osteosarcoma tissues or adjacent nontumor tissues. MTT, clone formation, wound healing, Transwell, in vivo tumorigenesis, flow cytometry and western blot analysis were used to determine cell proliferation, clone formation ability, migration, invasion, tumorigenesis, and cell apoptosis in MG63 and U2OS cells, respectively. Immunoprecipitation and immunofluorescence assays were performed to investigate the protein-protein interaction between HIF-1α and proteins related to signal pathways. HIF-1 was overexpressed in osteosarcoma tissues and cell lines, which promoted cell proliferation, clone formation, migration, invasion and inhibited cell apoptosis. Results also demonstrated that HIF-1 combined with AKT, and there might be a positive loop between the two proteins of HIF-1 and AKT, then the protein-protein interaction up-regulated the expression of Cyclin D1 in protein level, but not mRNA level, made Cyclin D1 protein more stable, triggered cell proliferation, clone formation, tumorigenesis, but inhibited cell apoptosis. The present study showed that HIF-1 modulated Cyclin D1 expression might through shaping a positive loop with AKT proteins. Additionally, HIF-1α promoted the tumor cells growth, migration and invasion in osteosarcoma through the activation of the AKT/Cyclin D1 signal cascade. We proposed that HIF-1 could be served as a marker for distinguishing osteosarcoma and an effective therapeutic target for osteosarcoma.

LncRNA WWOX‑AS1 inhibits the proliferation, migration and invasion of osteosarcoma cells.

Recently, numerous long non-coding (lnc)RNAs have been revealed as serving important roles in human gene regulation. Previous studies have suggested that aberrant expression of lncRNAs is associated with cancer progression and metastasis. Previous studies have also demonstrated that decreased expression of WW domain-containing oxidoreductase (WWOX) is associated with poor prognosis in numerous cancer types. However, the effect of WWOX antisense RNA 1 (WWOX-AS1) in the development of cancer remains unknown. The aim of the present study was to investigate the role of WWOX-AS1 in osteosarcoma. The expression levels of WWOX-AS1 in human osteosarcoma cell lines and a normal osteoblastic cell line were investigated using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The results revealed that WWOX-AS1 expression was downregulated in osteosarcoma tissues. Furthermore, the association between WWOX-AS1 and the prognosis of patients with osteosarcoma was investigated using Kaplan-Meier and log-rank tests. The results suggested that patients exhibiting high WWOX-AS1 expression demonstrated a greater overall survival compared with patients exhibiting low WWOX-AS1 expression. In addition, overexpression and knockdown of WWOX-AS1 was performed using transfection experiments and confirmed by RT-qPCR in MG63 and SAOS2 cells, respectively. The results demonstrated that WWOX-AS1 and WWOX expression were positively correlated. Furthermore, the results of the knockdown and overexpression functional experiments suggested that WWOX-AS1 overexpression inhibited the proliferation, migration and invasion of MG63 cells, and knockdown of WWOX-AS1 enhanced the proliferation, migration and invasion of MG63 cells in SAOS2 cells. In conclusion, the results of the present study suggested that WWOX-AS1 may represent a potential biomarker and therapeutic target for the treatment of osteosarcoma.

Rho A Regulates Epidermal Growth Factor-Induced Human Osteosarcoma MG63 Cell Migration.

Osteosarcoma, the most common primary bone tumor, occurs most frequently in children and adolescents and has a 5-year survival rate, which is unsatisfactory. As epidermal growth factor receptor (EGFR) positively correlates with TNM (tumor-node-metastasis) stage in osteosarcoma, EGFR may play an important role in its progression. The purpose of this study was to explore potential mechanisms underlying this correlation. We found that EGF promotes MG63 cell migration and invasion as well as stress fiber formation via Rho A activation and that these effects can be reversed by inhibiting Rho A expression. In addition, molecules downstream of Rho A, including ROCK1, LIMK2, and Cofilin, are activated by EGF in MG63 cells, leading to actin stress fiber formation and cell migration. Moreover, inhibition of ROCK1, LIMK2, or Cofilin in MG63 cells using known inhibitors or short hairpin RNA (shRNA) prevents actin stress fiber formation and cell migration. Thus, we conclude that Rho A/ROCK1/LIMK2/Cofilin signaling mediates actin microfilament formation in MG63 cells upon EGFR activation. This novel pathway provides a promising target for preventing osteosarcoma progression and for treating this cancer.

MicroRNA-183 suppresses the vitality, invasion and migration of human osteosarcoma cells by targeting metastasis-associated protein 1.

The aim of the present study was to investigate the effects of microRNA (miR)-183 on vitality, invasion, metastasis and apoptosis in osteosarcoma (OS) cells, mediated by its binding to metastasis-associated protein 1 (MTA1). A dual luciferase reporter assay was performed to determine whether MTA1 was a direct target of miR-183. Cell Counting Kit-8, Transwell, scratch-wound healing, fluorescence-activated cell sorting andterminal deoxynucleotidyl transferase dUTP nick end labeling assays were also performed to investigate the effects of miR-183 expression on the proliferation, invasion, migration and apoptosis of MG63 cells. It was demonstrated that that MTA1 expression levels were significantly higher in OS tissues and MG63 cells compared with corresponding adjacent noncancerous tissues and normal cells, respectively, while miR-183 expression levels were significantly lower (both P<0.05). Furthermore, miR-183 overexpression downregulated MTA1 levels and inhibited cell proliferation (P<0.05), migration (P<0.05) and invasion (P<0.01), as well as promoting apoptosis (P<0.01) by binding to the 3'-untranslated region of MTA1. These results indicate that miR-183 inhibits the vitality, invasion, migration and apoptosis of the OS cell line MG63 by targeting MTA1. These findings may contribute to the development of novel clinical therapeutic approaches for the treatment of OS.

ARTICLE WITHDRAWN] Dexmedetomidine Inhibits Osteosarcoma Cell Proliferation and Migration, and Promotes Apoptosis by Regulating miR-520a-3p.

This study aimed to investigate the effect of dexmedetomidine (DEX) on osteosarcoma (OS) cell line MG63 and to explore the possible relationship between DEX and miR-520-3p in OS. The results showed that DEX could upregulate miR-520-3p, which directly targeted AKT1. Additionally, miR-520-3p also inhibited MG63 cell proliferation and migration, promoted apoptosis, and suppressed protein expressions of AKT, p-AKT, p-mTOR, and p-ERK1/2. DEX can inhibit OS cell proliferation and migration and promote apoptosis by upregulating the expression level of miR-520a-3p. DEX may serve as a potential therapeutic agent in OS treatment, and miR-520a-3p may be a potential target in the therapy of OS.

Cyclic Guanosine Monophosphate (cGMP)-Dependent Protein Kinase II Blocks Epidermal Growth Factor (EGF)/Epidermal Growth Factor Receptor (EGFR)-Induced Biological Effects on Osteosarcoma Cells.

BackgroundThe present work was performed to detect the potential inhibitory effect of cyclic guanosine monophosphate (cGMP)-dependent protein kinase II (PKG II) on epidermal growth factor (EGF) receptor-induced biological activity and related signal cascades in osteosarcoma cells.Material/MethodsWe transfected the osteosarcoma MG-63 cell line with an adenoviral vector encoding PKG II cDNA (Ad-PKGII) and incubated the transfected cells with 250 μM 8-pCPT-cGMP to activate the PKG II. We stimulated the MG-63 cells with100 ng/ml EGF, and then detected their proliferation using a CCK-8 assay. Transwell assay was used to examine MG-63 cell migration; and Western blot analysis was used to detect expression of matrix metalloproteinase 9 (MMP-9) and activation of ERK and Akt.ResultsStimulating cells by 100 ng/ml EGF promoted MG-63 cell proliferation and migration, ERK and Akt phosphorylation, and MMP-9 expression. These effects of EGF were inhibited in MG-63 cells infected with Ad-PKGII and incubated with 8-pCPT-cGMP.ConclusionsOur results demonstrate that Ad-PKGII infection significantly inhibited EGF-induced proliferation and migration, as well as the associated-signal cascades; which indicates that PKG II might be a potential anti-cancer factor.

MicroRNA-338-3p inhibits proliferation, migration, invasion, and EMT in osteosarcoma cells by targeting activator of 90\xa0kDa heat shock protein ATPase homolog 1

BackgroundOsteosarcoma (OS) is a rare, malignant bone tumor that primarily affects adolescents and has a high degree of malignancy and high incidence of recurrence and metastasis. Our study aimed to explore the role of miR-338-3p in OS cells.MethodsqRT-qPCR was performed to quantify miR-338-3p expression levels in OS tissue samples and in three common OS cell lines. MG-63 and Saos2 cells were separately transfected with miR-338-3p or NC mimics and miR-338-3p expression levels was determined by qRT-PCR. Cell proliferation was monitored using the Cell Counting Kit-8. Flow cytometer analysis was carried out to determine the distribution of cell cycle stages and apoptosis. Transwell assay was performed to measure the migratory and invasive capacities of MG-63 and Saos2 cells. The expression of Vimentin and E-cadherin was detected by western blot. Luciferase reporter assay, qRT-PCR and western blotting were performed to confirm the target of miR-338-3p.ResultsAnalysis by qRT-PCR revealed that miR-338-3p was downregulated in the tissue samples of 20 OS patients when compared with that in their matched adjacent non-tumor tissues. Furthermore, miR-338-3p was significantly downregulated in three common OS cell lines, namely, MG-63, Saos2, and HOS, when compared with that in the human osteoblast cell line hFOB1.19. Analysis by luciferase reporter assay, qRT-PCR, and western blotting revealed that activator of 90 kDa heat shock protein ATPase homolog 1 (AHSA1) is a direct target of miR-338-3p. miR-338-3p overexpression led to significant reduction in AHSA1 protein levels in MG63 and Saos2 cells. miR-338-3p overexpression reduced cell viability and migration and invasion behavior of MG63 and Saos2 cells. In addition, miR-338-3p overexpression suppressed epithelial–mesenchymal transition (EMT), induced a significant G1-phase arrest and did not affect the apoptosis in both MG-63 and Saos2 cells. Moreover, overexpression of AHSA1 reversed the inhibitory effect of miR-338-3p overexpression on proliferation, cell cycle, apoptosis, EMT, migration, and invasion of MG63 and Saos2 cells, thereby suggesting that miR-338-3p acts as a tumor suppressor in OS cells by targeting AHSA1.ConclusionsmiR-338-3p/AHSA1 can serve as a potential therapeutic target for OS therapy.

GPNMB silencing suppresses the proliferation and metastasis of osteosarcoma cells by blocking the PI3K/Akt/mTOR signaling pathway

Glycoprotein non‑metastatic melanoma proteinB (GPNMB) is a glycoprotein that is highly expressed in various types of cancer, including osteosarcoma. However, its cellular functions and related mechanisms in osteosarcoma remain unclear. In the present study, a higher GPNMB mRNA level was observed in osteosarcoma tissues, than in adjacent non‑cancerous tissues. In addition, upregulation of the GPNMB mRNA and protein level was detected in the osteosarcoma cells SaOS2, 143B, MG63 and U2OS using western blot analysis and qPCR. Following transfection with GPNMB siRNA, the proliferation, migration and invasion of MG63 and U2OS cells were assessed using MTT and Transwell assays. The knockdown of GPNMB markedly inhibited the proliferation and metastasis of MG63 and U2OS cells. GPNMB silencing inhibited the activation of PI3K/Akt/mTOR signaling in MG63 and U2OS cells. PI3K/AKT activator insulin‑like growth factor‑1(IGF‑1) significantly activated the PI3K/Akt/mTOR signaling and reversed the suppressive effects of GPNMB silencing. IGF‑1 counteracted the inhibitory effects of GPNMB silencing on the proliferation and metastasis of the MG63 and U2OS cells. In conclusion, we provided evidence that GPNMB silencing regulated the proliferation and metastasis of osteosarcoma cells by suppressing the PI3K/Akt/mTOR signaling pathway. Thus, GPNMB may be a potential therapeutic target for osteosarcoma treatment.

MiR‑15a represses cancer cell migration and invasion under conditions of hypoxia by targeting and downregulating Bcl‑2 expression in human osteosarcoma cells.

Osteosarcoma is a common, high-risk primary bone malignancy that mostly affects the younger population. There has been no marked improvement in the clinical outcomes of osteosarcoma patients to date, and cancer recurrence and metastasis are common in high-grade osteosarcoma. Therefore, identifying new biomarkers and novel therapeutic targets is crucial for improving the prognosis of osteosarcoma patients. In the present study, the MG63 human osteosarcoma cell line was employed to examine the role of microRNA (miR)-15a in regulating cellular activities under hypoxic conditions. It was demonstrated that hypoxia stimulates migration and invasion in MG63 cells, which was correlated with the downregulation of miR-15a and upregulation of B-cell lymphoma 2 (Bcl-2) expression. Introduction of miR-15a or knockdown of endogenous Bcl-2 may reduce hypoxia-induced cell invasion and migration through the regulation of matrix metalloproteinases. Analysis of the expression of miR-15a indicated that hypoxia repressed the transcription of deleted in lymphocytic leukemia 2 (DLEU2), which is the host gene of miR-15a. These findings indicated that miR-15a may be a valuable target for the treatment of osteosarcoma, particularly for patients with high-grade cancer or heavy tumor burden.

WNT5A promotes migration and invasion of human osteosarcoma cells via SRC/ERK/MMP-14 pathway.

WNT5A, a representative ligand of activating several non-canonical WNT signal pathways, plays significant roles in oncogenesis and tumor inhibition. It has been shown that the non-receptor tyrosine kinase SRC is required for WNT5A-induced invasion of osteosarcoma cells. However, the precise molecular mechanism underlying WNT5A/SRC-mediated osteosarcoma cells invasion remains poorly defined. The study was designed to explore the role of ERK1/2 in WNT5A/SRC-induced osteosarcoma cells invasion and the downstream target of the SRC/ERK1/2 signalings. We found that WNT5A (100 ng/mL) remarkably stimulated migration and invasion of human osteosarcoma MG-63 cells, whereas inhibiting either SRC kinase activity by siRNA-mediated SRC silence or ERK1/2 phosphorylation by PD98059 treatment suppressed these effects, which suggested that the activation of SRC and ERK1/2 is essential for WNT5A-induced MG-63 cells migration and invasion. Furthermore, ERK1/2 phosphorylation induced by WNT5A was dramatically blocked by SRC siRNA. Additionally, our study further demonstrated that MMP-14 was upregulated after exposure to WNT5A in MG-63 cells, and the increased expression was blocked by SRC siRNA or PD98059. Collectively, these results indicate that WNT5A activates SRC/ERK1/2 signal pathway, leading to the upregulation of MMP-14 expression and MG-63 cells migration and invasion.

Osteostimulative calcium phosphosilicate biomaterials partially restore the cytocompatibility of decontaminated titanium surfaces in a peri-implantitis model.

Bacterial peri-implant biofilms, and the chemotherapeutics for their removal alter titanium surface cytocompatibility. In this study we aimed to assess the adjunctive use of an osteostimulative biomaterial utilizing a peri-implantitis model under the hypothesis that it will increase cell migration towards treated titanium surfaces. Acid-etched titanium surfaces were inoculated with a multi-species biofilm model and treated with 1.5% NaOCl in a previously characterized in vitro peri-implantitis model. Cell migration of MG63 cells towards the treated titanium surface (CTRL) was significantly reduced following inoculation with biofilm and chemotherapeutic treatment as compared to sterile controls. Addition of a tricalcium phosphate biomaterial (TCP) as a control for Ca+2 had a small non-significant effect, while BG significantly increased MG63 chemotaxis to titanium to levels comparable to sterile (STE). Similarly, cell viability at 5 days was increased in BG and TCP as compared to CTRL. SEM imaging confirmed the improved cytocompatibility of BG and TCP surfaces as compared to CTRL. Osteostimulative BG exhibited a strong chemotactic effect to osteoblasts, which was stronger than what was expected due to the chemotactic effect of Ca+2 alone (TCP). In addition, substantially increased cell attachment and viability was found on treated implant surfaces as compared to CTRL. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2645-2652, 2018.

MiR-183 inhibits osteosarcoma cell growth and invasion by regulating LRP6-Wnt/β-catenin signaling pathway

Recent studies have demonstrated that microRNA-183 (miR-183) deregulates and plays major roles in many tumors. However, the role of miR-183 in osteosarcoma (OS) pathogenesis is still largely unknown. In this study, we first over-expressed and knocked down miR-183 in MG63 and U20S cells, respectively. Functional analyses showed that ectopic expression of miR-183 suppressed MG63 cell growth, migration, and invasion in vitro and in vivo, whereas knockdown of endogenous miR-183 in U20S cells significantly enhanced these abilities. Next, we characterized low density lipoprotein receptor-related protein 6 (LRP6) as a direct target of miR-183 that interacted with the 3′-untranslated region of LRP6. Furthermore, ectopic expression of LRP6 significantly abrogated the tumor-suppressive effect induced by miR-183. Finally, miR-183 regulated the tumor-suppressive functions in MG63 cells by suppressing the LRP6-Wnt/β-catenin signaling pathway. Therefore, our study demonstrates that miR-183 is a tumor suppressor microRNA that plays a major role in OS.

LncRNA GAS5 Represses Osteosarcoma Cells Growth and Metastasis via Sponging MiR-203a

LncRNA GAS5, a growth suppressor, has been reported to exert anti-tumor actions in various cancers, whereas the exact mechanism underling the anti-tumor action is still unclear. This study was aimed to investigate the effect of lncRNA GAS5 on osteosarcoma and tried to decode the underling mechanisms. Expressions of lncRNA GAS5 in MG-63 cells were silenced by shRNA transfection, while were overexpressed by vector transfection. Cell viability, migration, invasion and apoptosis were respectively assessed by MTT, Transwell assay and flow cytometry. Regulations between lncRNA GAS5 and miR-203a, as well as between miR-203a and TIMP2 were detected by qPCR, western blot and dual luciferase activity assay. LncRNA GAS5 was down-regulated in MG-63 and OS-732 cells compared to hFOB1.19 cells. Silence of lncRNA GAS5 significantly promoted MG-63 cells viability, migration and invasion, and up-regulated Cyclin D1, Cyclin B1, CDK1 and CDK4 expressions. miR-203a was negatively regulated by lncRNA GAS5. The promoting activities of lncRNA GAS5 silence on MG-63 cells growth and metastasis were reversed by miR-203a suppression. TIMP2 was a target of miR-203a and the anti-growth and anti-metastasis actions of miR-203a suppression were reversed by TIMP2 silence. Further, lncRNA GAS5 silence, miR-203a overexpression, and TIMP2 silence could activate PI3K/AKT/GSK3β signaling while block NF-κB signaling. LncRNA GAS5 might be a tumor suppressor in osteosarcoma via sponging miR-203a, sequestering miR-203a away from TIMP2.

MiR-365 functions as a tumor suppressor by directly targeting CYR61 in osteosarcoma

Increasing evidence indicates that microRNAs(miRNAs) are often aberrantly expressed in osteosarcoma (OS) and play critical roles in OS tumorigenesis. Therefore, the discovery of miRNAs may provide a new and powerful tool for understanding the mechanismof OS initiation and development. The aim of this study was to investigate the functional significance of miR-365 and identify its possible mechanism in OS cells. Here, wefound that the expression level of miR-365 is significantly downregulated in OS tissues and cell lines, and its expression isassociated with the clinical stage, distant metastasis, tumor grade, and poor overall survival rate. The overexpression of miR-365 is able to inhibit cell proliferation, migration, and invasion in Saos-2 and MG-63 cells. Moreover, the cysteine-rich angiogenic inducer 61 (CYR61) has been identified as a target of miR-365 in OS cells, and its expression is found to be significantly increased in OS tissues, which is negatively correlated with miR-365. Furthermore, CYR61 overexpression significantly attenuated the suppressive effects of miR-365 on the proliferation, migration, and invasion of Saos-2 and MG-63 cells. Therefore, we consider that miR-365 acts as a tumor suppressor in OS, partly, by targeting CYR61 expression.

MicroRNA-505 is downregulated in human osteosarcoma and regulates cell proliferation, migration and invasion.

Recent studies have demonstrated that microRNAs (miRNAs/miRs) are involved in osteosarcoma tumorigenesis, progression, invasion and metastasis. For example, miR-505 plays important roles in human carcinogenesis; however, its exact function in osteosarcoma remains unclear. MicroRNA profiles of osteosarcoma and normal tissues were obtained by miRNA microarray assays, which were validated by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Then, high-mobility group box 1 (HMGB1) expression was evaluated by qRT-PCR and western blot analysis. The correlation between miR-505 and HMGB1 was analyzed by Pearson correlation. In vitro, the biological functions of miR-505 were examined by wound healing, MTT and Transwell assays and western blot analysis in MG63 cells transfected with miRNA mimics or empty vector. Luciferase assay was utilized to assess whether HMGB1 is a target of miR-505. miRNA microarrays revealed 26 aberrant miRNAs in osteosarcoma tissues; miR-505 showed the most pronounced decrease (P<0.01), which was significantly associated with TNM stage and metastasis status (P<0.05). In addition, HMGB1 was highly expressed in osteosarcoma tissues (P<0.01), with a significantly negative correlation with miR-505 (r=−0.6679, P<0.001). Furthermore, miR-505 inhibited proliferation, migration and invasion abilities of MG63 cells (P<0.01). Moreover, luciferase activity of the HMGB1-3′-UTR plasmid was suppressed following miR-505 binding (P<0.01). Finally, HMGB1 overexpression partly reversed the effects of miR-505 on MG63 cells. In conclusion, miR-505 levels are decreased in osteosarcoma tissues, and reduced miR-505 expression is significantly associated with poorer clinical prognosis in patients with osteosarcomas. miR-505 inhibits osteosarcoma cell proliferation, migration and invasion by regulating HMGB1.

RETRACTED: The effect of DNA-PKcs gene silencing on proliferation, migration, invasion and apoptosis, and in vivo tumorigenicity of human osteosarcoma MG-63 cells

The purpose of this study was to explore the role by which the DNA-dependent protein kinase complex catalytic subunit (DNA-PKcs) influences osteosarcoma MG-63 cell apoptosis, proliferation, migration and invasion. Osteosarcoma tissues and adjacent normal tissues were obtained from 57 osteosarcoma patients. Human osteosarcoma MG-63 cells were assigned into designated groups including the blank, siRNA-negative control (NC) and siRNA-DNA-PKcs groups. RT-qPCR and Western blotting methods were employed to evaluate the mRNA and protein expressions of DNA-PKcs. A cell counting kit-8 (CCK-8) assay was performed to assess cell viability. The evaluation of cell migration and invasion were conducted by means of Scratch test and Transwell assay. Flow cytometry with PI and annexin V/PI double staining was applied for the analysis of the cell cycle and apoptosis. Twenty-Four Balb/c nude mice were recruited and randomly divided into the blank, siRNA-NC and siRNA-DNA-PKcs groups. Tumorigenicity of the Balb/c nude mice was conducted to evaluate the rate of tumor formation, as well as for the assessment of tumor size and weight, and confirm the number of lung metastatic nodules in the mice post transfection. Osteosarcoma tissues were found to possess greater expression of DNA-PKcs than that of the adjacent normal tissues. DNA-PKcs expression in osteosarcoma tissues were correlated with the clinical stage and metastasis. Compared with the blank and siRNA-NC groups, proliferation, miration, as well as the invasion abilities of the MG-63 cells increased. Furthermore, an increase in apoptosis and cells at the G1 stage in the MG-63 cells was observed, while there were reductions in the cells detected at the S stage. The mRNA and protein expressions of CyclinD1, PCNA, Bcl-2 decreased while those of Bax increased in the siRNA-DNA-PKcs group. The tumor formation rate, tumor diameter, weight and lung metastatic nodules among the nude mice in the siRNA-DNA-PKcs group were all lower than those in the blank and siRNA-NC groups. The observations and findings of the study suggested that the silencing of DNA-PKcs inhibits the proliferation, migration and invasion, while acting to promote cell apoptosis in MG-63 cells and osteosarcoma growth in nude mice.

MiR-133b induces chemoresistance of osteosarcoma cells to cisplatin treatment by promoting cell death, migration and invasion.

As an important chemotherapeutic agent for the treatment of osteosarcoma, the effectiveness of cisplatin is considered to be due to its unique properties, which allow it to penetrate the cell membrane and form various DNA-platinum adducts, resulting in genetic alterations or DNA damage. However, chemoresistance to cisplatin remains a major challenge for its use and chemotherapeutic effects. In the present study, an isogenic model of a cisplatin resistant osteosarcoma cell line, MG63-DDP, was generated from the original MG63 cell line. The expression level of microRNA (miR)-133b in the MG63-DDP cisplatin-resistant osteosarcoma cell line was analyzed by reverse transcription-quantitative polymerase chain reaction (PCR). Cisplatin-DNA adduct formation, cell death (carboxyfluorescein succinimidyl ester/propidium iodide staining) and clonogenic survival assays (crystal violet staining) were performed, comparing various cell types. The effect of miR-133b on migration (scratch wound assay) and invasion (Transwell assay) was also evaluated. Characterization studies have previously revealed an increased level of miR-133b in MG63-DDP cells compared with normal MG63 cells. Upregulation of miR-133b was associated with the accumulation of cisplatin-DNA adducts and an increase in cisplatin-induced cell death. Furthermore, increased miR-133b expression levels enhanced the migration and invasion of MG63 cells under cisplatin stress. Concordantly, in MG63-DDP cells the neutralization of miR-133b demonstrated opposite effects, as compared with the upregulation of miR-133b. To the best of our knowledge, the present study demonstrated for the first time that cisplatin-resistant MG63 cells exhibit an increased level of miR-133b expression. The endogenous expression level of miR-133b is sufficient for inducing cisplatin resistance, which suggests that miR-133b may be a biomarker for cisplatin resistance in osteosarcoma.

Up-regulated lnc-SNHG1 contributes to osteosarcoma progression through sequestration of miR-577 and activation of WNT2B/Wnt/β-catenin pathway

Long noncoding RNA small nucleolar RNA host gene 1 (lnc-SNHG1) was reported to play an oncogenic role in the progression of cancers. However, the roles of SNHG1 and its molecular mechanism in osteosarcoma (OS) cells are largely unknown. In present study, we found that the expression of SNHG1 was up-regulated in OS tissues and cell lines. OS patients with the high SNHG1 expression were positively correlated with tumor size, TNM stage and lymph node metastasis. In addition, SNHG1 overexpression promoted cell proliferation, cell migration and EMT process in U2OS and MG63 cells and tumor growth in vivo. Furthermore, we also found that miR-577 could act as a ceRNAof SNHG1 in OS cells and the promotion of OS progression induced by lnc-SNHG1 overexpression required the inactivity of miR-577. Besides, we identified that WNT2B acted as a target of miR-577, and WNT2B played the oncogenic role in OS cells by activating Wnt/β-catenin pathway. In short, our study suggested that lnc-SNHG1 could promote OS progression via miR-577 and WNT2B. The lnc-SNHG1/miR-577/WNT2B/Wnt/β-catenin axis regulatory network might provide a potential new therapeutic strategy for OS treatment.

Efficacy of vitamins E and C for reversing the cytotoxic effects of nicotine and cotinine.

Nicotine has adverse cellular and molecular effects on oral mucosa, bone, and teeth. Vitamin E (α-tocopherol) and vitamin C (ascorbic acid) are biological antioxidants with positive effects on wound healing and bone formation. This in vitro study sought to assess the cytotoxic effects of different concentrations of nicotine and cotinine (a metabolite of nicotine) on MG-63 osteoblast-like cells and human gingival fibroblasts (HGFs) in the presence and absence of antioxidant vitamins E and C (separately and combined). Cell viability and proliferation were assessed using the methyl thiazol tetrazolium (MTT) assay. Cell migration was assessed using the scratch test, and expression of apoptosis-related genes was quantitatively analyzed using real-time PCR. Dose-dependent negative effects of nicotine on the morphology, viability, proliferation, and migration of MG-63 and HGF cells were statistically significantly greater than those of cotinine. Vitamin E (separately and combined with vitamin C) was statistically significantly more effective than vitamin C (at the concentration used in this study) at improving cell viability, proliferation, and migration, and at reducing apoptosis of cells exposed to nicotine or cotinine. Based on the positive results of this study, vitamin C and especially vitamin E (systemically and/or locally) may be useful in the repair and regeneration of oral hard and soft tissues in smokers.