Migration Of MG-63 Cells Research Articles

DDX10 overexpression predicts worse prognosis in osteosarcoma and its deletion prohibits cell activities modulated by MAPK pathway

Osteosarcoma (OS) is an invasive cancer in the skeletal system. The molecular mechanism of its etiology and pathogenesis are still not clear, so the effective treatment strategy of OS needs further research. First, we analyzed the expression level and prognostic ability of the RNA helicase DDX10 in OS patients based on the data obtained from GEO database. Next, we used CCK8 to test OS cell viability. Besides, we used wound-healing assay and transwell migration assay to detect cell migration of OS MG63 cell line. And the cell invasion was tested by transwell invasion assay. Moreover, we used QRT-PCR and western blot to analyze the mRNA and protein expression levels. We found that DDX10 was significantly over-expressed in OS patients and elevated level of DDX10 was associated with a poor prognosis. Silencing of DDX10 inhibited proliferation, invasion and migration of MG63 cells in vitro. Down-regulation of DDX10 inhibited MAPK signaling pathway. The expression of p-MEK and p-ERK were also decreased by silencing of DDX10. Therefore, Silencing of DDX10 inhibited proliferation, invasion and migration of MG63 cells, which might be regulated by suppression of MAPK pathway. In conclusion, our results unfold a novel area of studying for understanding how DDX10 functions in OS oncogenic and prognostic significance, accordingly implying a promising therapeutic target for OS treatment.

MiR-363-3p Inhibits Osteosarcoma Cell Proliferation and Invasion via Targeting SOX4.

miR-363-3p has been shown to suppress tumor growth and metastasis in various human cancers. However, the function of miR-363-3p in osteosarcoma (OS) has not been determined. In our study, we found that the expression of miR-363-3p was significantly downregulated in OS tissues compared with adjacent normal tissues. miR-363-3p expression was associated with the poor overall survival rate of OS patients. Moreover, we found that overexpression of miR-363-3p markedly inhibited the proliferation, migration, and invasion of U2OS and MG63 cells. Moreover, we found that SOX4 was a direct target of miR-363-3p in OS cells. Overexpression of miR-363-3p significantly inhibited the expression of SOX4. Expression levels of miR-363-3p and SOX4 were negatively correlated in OS tissues. Finally, we found that restoration of SOX4 attenuated the suppressive effects of miR-363-3p on the proliferation, migration, and invasion of U2OS and MG63 cells. Therefore, our findings demonstrated that miR-363-3p served as a tumor suppressor in OS tissues by targeting SOX4.

MicroRNA-185 inhibits proliferation, migration and invasion in human osteosarcoma MG63 cells by targeting vesicle-associated membrane protein 2

MicroRNAs (miRNAs) play an essential role in cancer development. Several studies have indicated that miRNAs mediate tumorigenesis processes, such as inflammation, proliferation, apoptosis and invasion. In the present study, we aimed to investigate the role of the microRNA-185 (miR-185) on the proliferation, migration and invasion of osteosarcoma (OS). MiR-185 expression was reduced in OS tissues and OS cell lines. MiR-185 inhibited OS cell proliferation, migration and invasion. Furthermore, a dual-luciferase assay validated that vesicle-associated membrane protein 2 (VAMP2) was a direct target of miR-185. Overexpression of miR-185 in OC cells reduced VAMP2 expression in protein and mRNA levels, whereas suppression of miR-185 led to an increase in VAMP2 protein and mRNA levels. In addition, we found that VAMP2 silencing inhibited the OS cell proliferation, migration and invasion. Further studies verified that introducing VAMP2 mRNA into cells over-expressing miR-185 abrogated the effects of miR-185 on OS cell proliferation, migration and invasion. Therefore, these results confirm that decreased expression of miR-185 might be regarded as a tumor marker for the early diagnosis of OS, by manipulating of its interactive factors with VAMP2, to provide an effective novel therapeutic target for treatment of the OS tumor.

DEAD-Box Helicase 5 Interacts With Transcription Factor 12 and Promotes the Progression of Osteosarcoma by Stimulating Cell Cycle Progression.

Osteosarcoma (OS) is a common malignant primary bone tumor. Its mechanism of development and progression is poorly understood. Currently, there is no effective therapeutic regimens available for the treatment of OS. DEAD-box helicase 5 (DDX5) is involved in oncogenic processes. This study aimed to explore the role of DDX5 in the development and progression of OS and its relationship with transcription factor 12 (TCF12), which is as an important molecule of Wnt signaling pathway. We found that the expressions of DDX5 and TCF12 protein were significantly higher in OS patients tissues and in the MG63 cells than in the corresponding normal tissues and human osteoblast cell hFOB 1.19. Overexpressions of both DDX5 and TCF12 were associated with clinicopathological features and poor prognosis of OS patients. siRNA based knockdown of DDX5 inhibited the proliferation of MG63 cells as demonstrated by an in vitro MTS assay and 5-ethynyl-2-deoxyuridine DNA proliferation detection, and promoted apoptosis of MG63 cells measured by flow cytometry. In addition, DDX5 knockdown inhibited the MG63 cell migration and invasion on transwell assays. Further experiments showed that DDX5 knockdown not only inhibited the expression of TCF12 but also decreased the mRNA and protein levels of Cyclin E1, an important regulator of G1–S phase progression, suggesting that DDX5 was required for the entry of cells into S phase. Overexpression of TCF12 reversed the cell proliferation, migration and invasion in MG63 cells induced by DDX5 knockdown accompanied by the upregulation of Cyclin E1. Additionally, we observed that DDX5 interacted with TCF12 in both OS tissues and MG63 cells by Co-immunoprecipitation assays. Taken together, our study revealed that DDX5 interacts with TCF12 and promotes the progression of OS by stimulating cell cycle progression. Our results suggest that DDX5 and TCF12 could be potential biomarkers for the diagnosis and treatment of OS.

LncRNA Linc00511 promotes osteosarcoma cell proliferation and migration through sponging miR-765.

Long noncoding RNA (lncRNA) Linc00511 is a novel lncRNA, and it was reported to play important roles in the progression and carcinogenesis of several tumors. However, the expression and biological roles of Linc00511 in osteosarcoma were still unknown. In this research, we showed that the expression of Linc00511 was upregulated in osteosarcoma samples and cell lines. Ectopic expression of Linc00511 promoted osteosarcoma cell growth, colony formation, and migration. Moreover, overexpression of Linc00511 enhanced the epithelial-mesenchymal transition progression in osteosarcoma cell. In addition, we showed that elevated expression of Linc00511 suppressed microRNA-765 (miR-765) expression and promoted apurinic/apyrimidinic endonuclease 1 (APE1) expression in osteosarcoma cell. The expression of miR-765 was downregulated in osteosarcoma cells and samples and was negatively related to Linc00511 expression in osteosarcoma tissues. Ectopic expression of miR-765 inhibited osteosarcoma cell growth and migration. Furthermore, we showed that Linc00511 overexpression promoted MG-63 cells proliferation, colony formation, and migration via downregulation of miR-765. These results suggested that Linc00511 played as an oncogene in the development of osteosarcoma.

Anti-tumoral potential of MDA19 in human osteosarcoma via suppressing PI3K/Akt/mTOR signaling pathway.

Osteosarcoma (OS) is the most common primary malignancy of skeleton with higher mortality rates amongst children and young adults worldwide, whereas effective and secure therapies have also been sought by researches with ongoing efforts. The purpose of the present study was to investigate the impact of N′-[(3Z)-1-(1-hexyl)-2-oxo-1,2-dihydro-3H-indol-3-ylidene] benzohydrazide (MDA19) on OS and explore its potential mechanism. Cell Counting Kit-8 (CCK8) and colony formation assay were employed to evaluate the potential effect of MDA19 on U2OS and MG-63 cells proliferation. Moreover, transwell migration and invasion assay were performed to assess the influence of MDA19 on U2OS and MG-63 cells migration and invasion. In addition, Annexin V-FITC/propidium iodide (Annexin V-FITC/PI) staining and flow cytometry were used to examine apoptotic ratio of the U2OS and MG-63 cells. Meanwhile, Western blot analysis was applied to explore change of relevant mechanism proteins in OS cells treated with MDA19. Our study showed that MDA19 had anti-proliferative activity of OS cells in a dose- and time-dependent manner, simultaneously, inhibition of colony formation was also observed in U2OS and MG-63 cells after incubation of MDA19. Besides, MDA19 could significantly inhibit the number of migrated and invaded OS cells and markedly increase the OS cells apoptosis rate. Mechanistically, we detected detectable reductions in apoptosis related proteins, epithelial–mesenchymal transition (EMT)-related proteins and activity of phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling in U2OS and MG-63 cells exposure to MDA19. Overall, the current study indicates in vitro anti-proliferative, anti-metastatic, and pro-apoptotic potential of MDA19 in U2OS and MG-63 cells. Our findings propose a clue for further studies with this compound in preclinical and clinical treatment for OS.

MicroRNA-885-5p promotes osteosarcoma proliferation and migration by downregulation of cell division cycle protein 73 homolog expression.

Osteosarcoma (OS) is the most common primary malignant bone tumor. Numerous studies have strongly implicated the ectopic expression of microRNAs (miRNAs/miRs), including miR-885-5p, which is aberrantly expressed in several cancer types, in multiple cancer-related processes. However, the role of miR-885-5p in OS remains unknown. In the present study, it was found that the expression of miR-885-5p was markedly upregulated in OS cell lines and clinical tissues. Moreover, high expression of miR-885-5p was significantly associated with the development of OS. The human OS MG-63 cell line was transfected with recombinant lentivirus to regulate miR-885-5p expression. Overexpressed miR-885-5p significantly promoted the proliferation and migration of MG-63 cells in vitro, while downregulating miR-885-5p expression reversed these effects. Furthermore, bioinformatic analysis was used to predict the potential target genes of miR-885-5p, and cell division cycle protein 73 homolog (CDC73) was identified as a novel and direct target of miR-885-5p. This interaction was further confirmed using reverse transcription-quantitative polymerase chain reaction, western blotting and luciferase activity assays. These findings suggest that miR-885-5p serves a critical role in facilitating OS proliferation and migration, and can regulate CDC73 expression in OS cells and tissues. Thus, miR-885-5p could be a promising novel therapeutic biomarker for OS.

Astragaloside IV promotes the proliferation and migration of osteoblast-like cells through the hedgehog signaling pathway.

The present study aimed to investigate the effects of astragaloside IV on osteoblast-like cell proliferation and migration, in addition to the underlying signaling pathway. In order to observe the effect on proliferation, a Cell Counting Kit-8 assay and flow cytometry were used. To detect cell migration ability, cell scratch and Transwell cell migration assays were performed. The RNA and protein expression levels of hedgehog signaling molecules, including Sonic hedgehog (SHH) and GLI family zinc finger 1 (GLI1), were examined by reverse transcription-quantitative polymerase chain reaction and western blot analyses. To inhibit the hedgehog signaling pathway, cyclopamine was used. Astragaloside IV, at a dosage of 1×10−2 µg/ml in MG-63 cells and 1×10−3 µg/ml in U-2OS cells, resulted in the enhanced proliferation and migration of cells, and the gene expression levels of the SHH and GLI1 were significantly increased. The combination of astragaloside IV and cyclopamine reduced MG-63 and U-2OS cell proliferation and migration, and inhibited the gene expression of SHH and GLI1. Astragaloside IV enhanced the proliferation and migration of human osteoblast-like cells through activating the hedgehog signaling pathway. The results of the present study provide a rational for the mechanistic link in astragaloside IV promoting the proliferation and migration of osteoblasts via the hedgehog signaling pathway.

MALAT1 promotes proliferation, migration, and invasion of MG63 cells by upregulation of TGIF2 via negatively regulating miR-129.

PurposeThis article aimed to investigate the mechanism by which MALAT1 and miR-129 affected the development of osteosarcoma.MethodsTumor tissues and adjacent tissues of 23 osteosarcoma patients were collected. Normal osteoblasts hFOB1.19 and osteosarcoma cells MG63 were cultured. MG63 cells were transfected and grouped: si-negative control (NC) group, si-MALAT1 group, miR-129 NC group, miR-129 mimics group, p-Empty vector group, p-MALAT1 group, p-MALAT1+ miR-129 mimics group, and p-MALAT1+ si-TGIF2 group. Luciferase reporter assay, Cell Counting Kit-8 assay, Transwell assay, quantitative reverse transcription PCR, Western blot, and Pearson correlation analysis were performed.ResultsMALAT1 expression in tumor tissues and MG63 cells was increased (P<0.01). High MALAT1 expression predicted poor prognosis of osteosarcoma patients. MG63 cells of si-MALAT1 group exhibited much lower cell viability, migration, and invasive cell numbers when compared with si-NC group (P<0.01). For MG63 cells of miR-129 mimics group, they had markedly lower cell viability, migration, and invasive cell numbers than miR-129 NC group (P<0.01). miR-129 was targetedly and negatively regulated by MALAT1. TGIF2, which was targetedly and negatively regulated by miR-129, was overexpressed in tumor tissues and MG63 cells (P<0.01). miR-129 overexpresison and TGIF2 downregulation significantly reversed the enhanced cell viability, migration, and invasion induced by MALAT1 (P<0.01).ConclusionMALAT1 promotes TGIF2 expression through negative regulation of miR-129, which further promotes the proliferation, migration, and invasion of MG63 cells.

Proliferation inhibition and apoptosis promotion by dual silencing of VEGF and Survivin in human osteosarcoma.

Simultaneous silencing of multiple upregulated genes is an attractive and viable treatment strategy for many incurable diseases including cancer. Herein we used a dual gene-targeted siRNA conjugate composed of VEGF and Survivin siRNA sequences in the same backbone to inhibit proliferation and angiogenesis in two human osteosarcoma cell lines. We synthesized siRNA sequences targeting the VEGF and Survivin genes individually (VEGF siRNA and Survivin siRNA) or simultaneously (one-chain-double-target siRNA: dual siRNA). VEGF and Survivin mRNA and protein expression levels in human osteosarcoma MG-63 and Saos-2 cells were detected by qRT-PCR and western blot analysis. VEGF and Survivin protein location and expression were evaluated by immunohistochemistry and immunofluorescence staining. MG-63 and Saos-2 cell migration, proliferation, apoptosis, and angiogenesis were detected by scratch test, MTT assay, flow cytometry, and capillary tube assay respectively. The dual siRNA induced similar downregulation of VEGF and Survivin mRNA and protein levels, compared with VEGF siRNA or Survivin siRNA alone. The dual siRNA caused greater suppression of MG-63 and Saos-2 cell migration, proliferation and angiogenesis, and promoted more cell apoptosis than VEGF siRNA or Survivin siRNA alone, suggesting that the effects of the dual siRNA on inhibiting cell proliferation, migration, and angiogenesis and promoting apoptosis were superior to those of the single-target siRNAs. Simultaneous silencing of VEGF and Survivin using the dual siRNA may be an advantageous alternative for the development of therapeutic strategies against human osteosarcoma.

Indole-3-Carbinol Induces Apoptosis in Human Osteosarcoma MG-63 and U2OS Cells.

This study was focused on investigating the anticancer potential of indole-3-carbinol (I3C) against osteosarcoma MG-63 and U2OS cells. A wound healing assay indicated that IC3 inhibited migration of MG-63 and U2OS cells. MTT, WST-1, and colony formation assays revealed that treatment of MG-63 and U2OS cells with I3C decreased cell viability. Fluorescence-activated cell sorting (FACS) analysis showed that I3C induced apoptosis in a dose- and time-dependent manner in MG-63 and U2OS cells. Moreover, via terminal deoxynucleotidyl transferase- (TdT-) mediated dUTP-biotin nick-end labeling (TUNEL) assay, we detected that I3C induced DNA fragmentation. Western blotting demonstrated that activated forms of caspase-3, caspase-7, and caspase-9, as well as poly (ADP-ribose) polymerase (PARP) were increased in MG-63 and U2OS cells, following treatment with I3C. Furthermore, protein expression levels of FOXO3, Bax, and Bim extra-large form were increased while those of Akt, JNK, p38, phosphorylated ERK, and Bcl-xL were decreased by I3C treatment in MG-63 and U2OS cells. Thus, the study indicates that I3C may induce apoptosis in human osteosarcoma MG-63 and U2OS cells via the activation of apoptotic signaling pathways by FOXO3.

Synergistic anticancer effects of timosaponin AIII and ginsenosides in MG63 human osteosarcoma cells

BackgroundTimosaponin AIII (TA3) is a steroidal saponin extracted from Anemarrhena asphodeloides. Here, we investigated the anticancer effects of TA3 in MG63 human osteosarcoma cells. TA3 attenuates migration and invasion of MG63 cells via regulations of two matrix metalloproteinases (MMPs), MMP-2 and MMP-9, which are involved with cancer metastasis in various cancer cells. TA3 reduced enzymatic activities and transcriptional expressions of MMP-2 and MMP-9 in MG63 cells. TA3 also inhibited Src, focal adhesion kinase, extracellular signal-regulated kinase (ERK1/2), c-Jun N-terminal kinase (JNK), p38, β-catenin, and cAMP response element binding signaling, which regulate migration and invasion of cells. TA3 induced apoptosis of MG63 cells via regulations of caspase-3, caspase-7, and poly(ADP-ribose) polymerase (PARP). Then, we tested several ginsenosides to be used in combination with TA3 for the synergistic anticancer effects. We found that ginsenosides Rb1 and Rc have synergistic effects on TA3-induced apoptosis in MG63 cells. MethodsWe investigated the anticancer effects of TA3 and synergistic effects of various ginseng saponins on TA3-induced apoptosis in MG63 cells. To test antimetastatic effects, we performed wound healing migration assay, Boyden chamber invasion assays, gelatin zymography assay, and Western blot analysis. Annexin V/PI staining apoptosis assay was performed to determine the apoptotic effect of TA3 and ginsenosides. ResultsTA3 attenuated migration and invasion of MG63 cells and induced apoptosis of MG63 cells. Ginsenosides Rb1 and Rc showed the synergistic effects on TA3-induced apoptosis in MG63 cells. ConclusionsThe results strongly suggest that the combination of TA3 and the two ginsenosides Rb1 and Rc may be a strong candidate for the effective antiosteosarcoma agent.

Von Willebrand factor rescued by miR-24 inhibition facilitates the proliferation and migration of osteosarcoma cells in vitro.

von Willebrand factor (vWF) is a major procoagulant molecule that was shown to differentiate between metastatic and primary osteosarcoma (OS) tissues and associated with increased metastasis. However, its functional role in OS progression has been unclear yet. The expression profile of vWF and miR-24 in human OS tissues was characterized using immunofluorescence labeling and quantitative real-time PCR analysis. The interaction between miR-24 and vWF was identified by dual luciferase reporter assay. The effects of vWF and miR-24 on OS cells were assessed by cell proliferation, colony formation, and migration. The clinical significance of miR-24 in OS patients was analyzed using Kaplan–Meier analyses and Pearson’s Chi-squared test. Here, we reported that the expression of vWF was significantly increased, but miR-24 was significantly decreased in OS tissues (n=84). vWF was further validated as the target of miR-24 in MG-63 and U2OS cells. miR-24 obviously suppressed the proliferation and migration of MG-63 and U2OS cells. However, the migration-inhibiting activity of miR-24 was predominantly attenuated by vWF overexpression. Clinically, low miR-24 expression in human OS tissues was significantly associated with tumor metastasis and predicted a poor survival in OS patients. This work demonstrated that vWF, as a downstream effector of miR-24, played an important role in controlling OS cell progression. Target miR-24 or vWF, therefore, promises to be an effective biological target for OS treatment.

TUFT1 promotes osteosarcoma cell proliferation and predicts poor prognosis in osteosarcoma patients

ObjectiveThis study is aimed at exploring the role of TUFT1 in osteosarcomas.MethodsWe investigated the expression of TUFT1 in osteosarcoma cell lines and explored the correlation between TUFT1 expression and prognosis in osteosarcoma patients based on the expression data downloaded from Gene Expression Omnibus (GEO) website. The effects of TUFT1 on osteosarcoma cell proliferation, migration and invasion were investigated by silencing TUFT1 in osteosarcoma MG63 cell line. Finally, western blot was performed to determine the expression changes of MAPK signaling pathway related proteins after silencing TUFT1.ResultsWe found that the expression of TUFT1 was significantly up-regulated in osteosarcoma cell lines compared with the normal control. Using Kaplan-Meier analysis, we identified that high TUFT1 expression was positively correlated with poor prognosis in osteosarcoma patients. Furthermore, knockdown of TUFT1 remarkably inhibited MG63 cell proliferation, migration and invasion. Using western blot analysis, we found that the phosphorylation levels of MEK and ERK were reduced obviously in MG63 cells after silencing TUFT1 (p<0.01).ConclusionsOur results demonstrated that TUFT1 plays a promoting role in MG63 cell proliferation and metastasis and has the potential to be a predictor as well as a therapeutic target for osteosarcoma patients.

Long Noncoding RNA FEZF1-AS1 Promotes Osteosarcoma Progression by Regulating the miR-4443/NUPR1 Axis.

Long noncoding RNA (lncRNA) FEZF1-AS1 was demonstrated to facilitate cell proliferation and migration in some cancers. However, the functions of FEZF1-AS1 and its molecular mechanism in osteosarcoma remain to be elucidated. In our study, we found that the expression of FEZF1-AS1 was upregulated in osteosarcoma samples and cell lines compared with normal tissues or cells. Besides, we showed that the expression levels of FEZF1-AS1 in osteosarcoma patients were positively correlated with tumor metastasis and TNM stage. Additionally, FEZF1-AS1 knockdown inhibited cell proliferation, migration, and invasion in U2OS and MG63 cells, while upregulation had the opposite effects in vitro. Moreover, FEZF1-AS1 depletion inhibited tumor growth and metastasis in vivo. We found that FEZF1-AS1 sponged miR-4443 to promote NUPR1 expression in U2OS and MG63 cells. Furthermore, knockdown of miR-4443 abrogated FEZF1-AS1 silencing-induced inhibition of cell proliferation, migration, and invasion in osteosarcoma. Finally, we found that restoration of NUPR1 rescued the proliferation, migration, and invasion abilities of FEZF1-AS1-depleted U2OS and MG63 cells. Our study indicated that FEZF1-AS1 could promote osteosarcoma progression by sponging miR-4443 to promote NUPR1 expression. The FEZF1-AS1/miR-4443/NUPR1 axis may act as a novel therapeutic strategy for osteosarcoma treatment.

MiR-216b inhibits osteosarcoma cell proliferation, migration, and invasion by targeting Forkhead Box M1.

Osteosarcoma (OS) is considered the most common type of primary malignant bone tumor, which has a high rate of mortality in children and adolescents. However, the current treatment methods for OS are ineffective. Therefore, there is an urgent requirement to identify the critical targets. This study aimed to identify the roles and significance of microRNA-216b (miR-216b) in OS. To explore the cellular and molecular functions of miR-216b and Forkhead Box M1 (FoxM1) in OS, the expression of miR-216b and FoxM1 at the transcriptional level was measured using quantitative real-time PCR (qRT-PCR). Wound healing assay, 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide assay (MTT) assay, flow cytometry, and transwell invasion assay were conducted to study the function of miR-216b and FoxM1 in OS cells. Dual luciferase reporter assay was performed to identify the relationships between miR-216b and FoxM1. qRT-PCR results revealed that miR-216b expression was significantly downregulated, and FoxM1 was observed to be significantly upregulated in human OS cell lines (MG-63) and tissues. MTT data showed that upregulation of miR-216b expression led to cell growth inhibition in MG-63 cells. The results of the invasion assay and wound healing assay illustrated that miR-216b upregulation or FoxM1 downregulation could inhibit the invasion and migration in MG-63 cells. In vivo, the tumor volume was significantly decreased by miR-194 mimic treatment compared with the control group. Furthermore, the results of the luciferase assay indicated that FoxM1 is a direct target of miR-216b. These findings may provide novel insights into the molecular mechanism of miR-216b and FoxM1 in the progression of OS, and suggested that miR-216b may serve as a potential tumor inhibitor of OS by targeting FoxM1.

Oridonin prevents epithelial-mesenchymal transition and TGF-β1-induced epithelial-mesenchymal transition by inhibiting TGF-β1/Smad2/3 in osteosarcoma

Osteosarcoma is the most common primary bone tumor with highly invasive characteristic and low long-term survival. Recently, epithelial-mesenchymal transition (EMT) is reported as a key event in cancer invasion and metastasis. Oridonin, a bioactive diterpenoid, has been proved to possess anti-cancer effects. However, the effect of oridonin on EMT and metastasis of osteosarcoma is unclear. In this study, we investigated the underlying mechanism of oridonin on EMT and metastasis of osteosarcoma. We found that oridonin inhibited migration and invasion of MG-63 and 143B cells. Moreover, oridonin increased the protein expression of E-cadherin and decreased that of N-cadherin and Vimentin. Oridonin upregulated the transcription of E-cadherin and downregulated N-cadherin and Vimentin. Oridonin inhibited the protein and mRNA levels of Snail and Slug. Furthermore, oridonin inhibited TGF-β-induced phosphorylation of Smad 2/3, prevented Smad dimer translocation into the nucleus. Finally, we established metastatic models of osteosarcoma 143B cells, and found that oridonin inhibited lung metastasis in vivo. Oridonin increased the protein expression of E-cadherin and reduced N-cadherin and Vimentin. Oridonin inhibited the protein expression of Snail and Slug as well as Smad 2/3 activation. In conclusion, our study demonstrated that oridonin inhibited EMT and TGF-β1-induced EMT by inhibiting TGF-β1/Smad2/3 signaling pathway in osteosarcoma.

Adenovirus-mediated small interfering RNA targeting ezrin induces apoptosis and inhibits metastasis of human osteosarcoma MG-63 cells.

Osteosarcoma is a disease prone to recurrence and metastasis, and adenovirus expression vector is frequently studied as a therapeutic target of osteosarcoma in recent years. The present study attempts to explore the effect of adenovirus-mediated siRNA targetting ezrin on the proliferation, migration, invasion, and apoptosis of human osteosarcoma MG-63 cells. Human osteosarcoma MG-63 cell line was selected for construction of recombinant adenovirus vector. The mRNA and protein levels of ezrin, Bcl2-associated X protein (Bax), B cell lymphoma-2 (Bcl-2), p21, p53, Caspase-3, matrix metalloproteinase (MMP) 2 (MMP-2) and MMP-9, Cyclin D1, and cyclin-dependent kinase 4a (CDK4a) were determined. Through ELISA, the levels of Caspase-3, MMP-2 and MMP-9 were examined. Finally, human osteosarcoma MG-63 cell viability, growth, invasion, migration, and apoptosis were detected. Initially, adenovirus expression vector of ezrin was constructed by ezrin 2 siRNA sequence. Adenovirus-mediated siRNA targetting ezrin reduced expression of ezrin in MG-63 cells. The results revealed that adenovirus-mediated siRNA targetting ezrin elevated expression levels of Bax, p21, p53, and Caspase-3, Cyclin D1, and CDK4a and reduced expression levels of Bcl-2, MMP-2 and MMP-9. Furthermore, adenovirus-mediated siRNA targetting ezrin inhibited human osteosarcoma MG-63 cell viability, growth, invasion, and migration, and promoted apoptosis. Our study demonstrates that adenovirus-mediated siRNA targetting ezrin can induce apoptosis and inhibit the proliferation, migration, and invasion of human osteosarcoma MG-63 cells.

MiR-30a-5p inhibits osteosarcoma cell proliferation and migration by targeting FOXD1

Despite a number of studies have emphasized the extensive role of microRNA (miRNA) in the development of multiple cancers, the role of miR-30a-5p in the progression of osteosarcoma (OS) and the underlying mechanism are still limited. We detected the expression level of MiR-30a-5p and forkhead box D1 (FOXD1) in Clinical OS specimens and found that miR-30a-5p was significantly decreased while FOXD1 was markedly increased. Dual luciferase assay confirmed that FOXD1 was directly regulated by miR-30a-5p. In vitro assay showed that inhibitior of FOXD1 suppressed cell proliferation, migration and invasion in MG63 and U2OS cells, while overexpression of FOXD1 promoted OS cell proliferation and migration. In vivo assay further showed the inhibition of tumor growth after knockdown of FOXD1. These results suggested that FOXD1 might play key roles in OS development and progression, and was negatively regulated by miR-30a-5p in OS.

MicroRNA-103 Promotes Proliferation and Inhibits Apoptosis in Spinal Osteosarcoma Cells by Targeting p57.

Osteosarcoma is one of the most aggressive malignancies with poor prognosis rates. Many studies have demonstrated that miRNAs were involved in osteosarcoma, but the role of miR-103a in osteosarcoma remains elusive. In this study, we detected the expression levels of miR-103 in osteosarcoma and non-osteosarcoma tissues and cell lines. The binding effect of miR-103 on p57 was detected by luciferase reporter assay. After altering expressions of miR-103 or p57, viability, migration, invasion, and apoptosis of MG63 cells and expressions of proteins related with the JNK/STAT and mTOR pathways were all detected. We found the higher expression of miR-103 in osteosarcoma tissues and cell lines compared with non-osteosarcoma tissues and cell lines. miR-103 overexpression promoted survival, migration, and invasion of MG63 cells. Knockdown of miR-103a inhibited cell survival, migration, and invasion by upregulating the expression of p57, which was a target of miR-103. Moreover, miR-103a overexpression activated the JNK/STAT and mTOR pathways probably through inhibiting p57 expression. In conclusion, miR-103a acted as an oncogene in osteosarcoma, probably through activating the JNK/STAT and mTOR pathways by inhibiting p57 expression.