Epithelial-mesenchymal Transition Of Osteosarcoma Cells Research Articles

Diallyl trisulfide inhibits osteosarcoma 143B cell migration, invasion and EMT by inducing autophagy

BackgroundDiallyl trisulfide (DATS), a compound derived from garlic, has been demonstrated its anti-cancer properties. While it has been shown to inhibit the expression of epidermal growth factor receptor (EGFR) in various cancers, its effects on osteosarcoma (OS) cells remain unclear. This study aimed to investigate the impacts of DATS on OS cells growth, migration, invasion, epithelial-mesenchymal transition (EMT) and autophagy, as well as its underlying mechanisms which was involving in the EGFR/PI3K/AKT/mTOR pathway. MethodsIn this study, human osteosarcoma cells (143B) were treated with different concentrations of DATS (10, 50, 100 and 200 μM) for 24 and 48 h, respectively. Cell viability was measured using CCK8, the half lethal concentration was selected for the following experiments. Wound healing and transwell assays were performed to evaluate migration and invasion abilities, while flow cytometry was used to measure apoptosis. Quantitative reverse transcription polymerase chain reaction (qRT-PCR), Western blotting, and confocal imaging were employed to analyze the related mRNA and protein expression levels of epithelial-mesenchymal transition (EMT), EGFR/Phosphoinositide 3 kinase (PI3K)/AKT/Mammalian target of rapamycin (mTOR) signaling pathway and autophagy-related markers. ResultsDATS significantly inhibited proliferation, migration and EMT in osteosarcoma cells. Additionally, DATS promoted cell apoptosis and induced autophagy, which could be rescued by the autophagy inhibitor 3-methyladenine (3-MA). Moreover, DATS treatment led to the inactivation of the EGFR/PI3K/AKT/mTOR pathway in osteosarcoma cells. ConclusionsThis study demonstrated that DATS inhibited osteosarcoma cell growth, migration and EMT, but inducing apoptosis and autophagy. These effects were mediated by the inactivation of the EGFR/PI3K/AKT/mTOR signaling pathway. These findings suggested that DATS could serve as a potential therapeutic agent for osteosarcoma treatment.

DLX2 promotes osteosarcoma epithelial-mesenchymal transition and doxorubicin resistance by enhancing HOXC8-CDH2 axis

Metastasis and doxorubicin resistance are challenges in the clinical diagnosis and treatment of osteosarcoma, the mechanisms underlying these phenomena remain unclear. In this study, we found that DLX2 is highly expressed in metastatic osteosarcoma and is closely related to clinical prognosis. Knockdown of DLX2 inhibited tumor proliferation and migration invitro and inhibited tumor growth invivo. Mechanistically, we found that DLX2 enhanced the repression of CDH2 transcription by binding to HOXC8, thereby promoting the epithelial-mesenchymal transition in osteosarcoma cells. Through subsequent exploration, we found that targeting DLX2/HOXC8 signaling significantly restores the sensitivity of osteosarcoma cells to doxorubicin. In conclusion, our findings demonstrate that DLX2 may enhance the transcriptional regulation of CDH2 through interacting with HOXC8, which in turn promotes epithelial-mesenchymal transition and doxorubicin resistance in osteosarcoma. These findings hold great potential for clinical application and may guide the development of novel targeted therapies for osteosarcoma.

The mechanism of VCP-mediated metastasis of osteosarcoma based on cell autophagy and the EMT pathway.

Study of the molecular mechanisms of metastasis is still the research focus for osteosarcoma (OS) prevention. This study investigates the mechanism of valosin-containing protein (VCP) promoting OS metastasis in vitro through autophagy and epithelial-mesenchymal transition (EMT). Different cell lines of osteosarcoma (143B and MG63) were adopted in this study. The level of VCP expression in osteosarcoma cells was changed, and the level of autophagy and the progression of the epithelial-mesenchymal transition (EMT) were observed. Then autophagy and EMT in OS cells were changed artificially, and proliferation and migration ability were observed. The expression of LC3II/I was decreased, but the insolubilized P62 protein expression was increased in the VCP inhibiting group and the autophagy inhibitor treatment group. Simultaneously, E-cadherin protein expression increased while N-cadherin protein expression decreased in the VCP inhibiting group but increased in the TGF-β1 treatment group. In addition, suppressing VCP can cause a decrease in Transforming Growth Factor β1 (TGF-β1), smad2, smad3, phosphorylated smad2 (p-smad2), and phosphorylated smad3 (p-smad3). Autophagy inhibitors and agonists have no significant effect on the migration and invasion of OS cells but can significantly affect the ability of cells to resist anoikis. EMT inhibitors and agonists have a proportional effect on the migration and invasion of OS cells. VCP is likely to promote the migration and invasion of OS cells by inducing EMT, possibly via TGF-β1/smad2/3 signaling pathway. In this process, VCP-mediated autophagy may contribute to successful distant metastasis of tumor cells indirectly.

Long non-coding RNA MALAT1 promotes cell proliferation, migration and invasion by targeting miR-590-3p in osteosarcoma.

Osteosarcoma (OS) is a common malignant bone cancer and commonly occurs in adolescents and children. Long non-coding RNAs (lncRNAs) play major roles in cancer cell proliferation and metastasis. The present study aimed to investigate the potential molecular mechanism of lncRNA MALAT1 in OS. The levels of lncRNA MALAT1 and microRNA-590-3p were detected by reverse transcription-quantitative PCR in OS tissues and cells. Cell Counting Kit-8 and flow cytometry assays were conducted to assess cell proliferation and apoptosis. Cell migration and invasion were examined by Transwell assay. The levels of E-cadherin, N-Cadherin, Vimentin and Snail were measured by western blotting. The target of MALAT1 was predicted using online software and confirmed by luciferase reporter, RNA immunoprecipitation and RNA pull-down assays. The results indicated that MALAT1 was highly expressed in OS tissues and cell lines. MALAT1 knockdown promoted apoptosis and suppressed proliferation, migration, invasion and epithelial- mesenchymal transition (EMT) of OS cells. Overexpression of miR-590-3p increased cell apoptosis and hampered cell proliferation, migration, invasion and EMT in OS cells. In addition, MALAT1 knockdown upregulated the expression of miR-590-3p in OS cells. In conclusion, MALAT1 was demonstrated to suppress cell apoptosis and induce cell proliferation, migration, invasion and EMT by inhibiting miR-590-3p in OS, which indicated that MALAT1 has potential value in the diagnosis and treatment of OS.

Propofol inhibits the malignant development of osteosarcoma U2OS cells via AMPK/FΟΧO1-mediated autophagy.

It has previously been reported that propofol regulates the development of human osteosarcoma (OS). However, the specific molecular mechanisms underlying the effect of propofol on OS remain poorly understood. Therefore, the aim of the present study was to explore the effects of propofol on OS U2OS cells and the potential underlying mechanism. The Cell Counting Kit-8 and colony formation assays were performed to assess cell viability and proliferation. Furthermore, cell apoptosis was assessed using the TUNEL assay and western blotting. Wound healing and Transwell assays were performed to evaluate OS cell migration and invasion abilities, respectively. The protein expression levels of epithelial-mesenchymal transition (EMT)-, autophagy- and adenosine monophosphate-activated protein kinase (AMPK)/FOXO1 signaling pathway-related proteins were also determined using western blotting. The results demonstrated that propofol significantly reduced the viability of OS cells and promoted autophagy in a dose-dependent manner. Moreover, cell treatment with propofol significantly enhanced the protein expression levels of phosphorylated (p)-AMPK and FOXO1, while decreasing the protein levels of p-FOXO1. Furthermore, treatment with propofol significantly suppressed cell viability, migration and invasion abilities and the EMT of OS cells, and potentially promoted cell apoptosis via inducing autophagy via the AMPK/FOXO1 signaling pathway. In summary, the present study indicated that propofol potentially had an inhibitory effect on the development of OS cells via AMPK/FOXO1-mediated autophagy. These results have therefore provided an experimental basis for further studies into the therapeutic effect of propofol on OS.

LINC01140 regulates osteosarcoma proliferation and invasion by targeting the miR-139-5p/HOXA9 axis

Osteosarcoma is one of the commonest metastatic tumor in children and teenagers, and has a hopeless, prognosis. Long non-coding RNA (lncRNA) acts momentous roles as a regulator on the proliferation and migration of cancer. Here, we performed GEO database analysis and qPCR to identify differentially expressed lncRNAs in osteosarcoma cells. Knockdown of lncRNA LINC01140 was used to detect the effect of LINC01140 on the proliferation, invasion, and epithelial-mesenchymal transition (EMT) of osteosarcoma cells. Bioinformatics analysis and qPCR identified the LINC01140/miR-139-5p/Homeobox A9 (HOXA9) regulatory axis. RNA immunoprecipitation assay, Dual-luciferase assay, and rescue experiments confirmed the interaction of LINC01140/miR-139-5p/HOXA9 in osteosarcoma. LINC01140 was overexpressed in osteosarcoma and knocking down LINC01140 restrained the proliferation and invasion of osteosarcoma cells and EMT. In Saos2 and MG63 cells, LINC01140 sponged miR-139-5p, and a miR-139-5p inhibitor overturned the suppression of LINC01140 knockdown on the proliferation and migration of osteosarcoma cells. Moreover, miR-139-5p depressed the invasion, proliferation, and EMT of osteosarcoma cells via targeting HOXA9. Our results indicate that LINC01140 downregulation inhibits the invasion, proliferation, and EMT in osteosarcoma cells through targeting the miR-139-5p/HOXA9 axis. Therefore, LINC01140 is a potential therapeutic target for osteosarcoma.

LncRNA CBR3-AS1 promotes osteosarcoma progression through the network of miR-140-5p/DDX54-NUCKS1-mTOR signaling pathway

Long noncoding RNA (lncRNA) CBR3-AS1 (termed as CBR3-AS1) has been reported to be upregulated in several cancers including osteosarcoma. Its positive impact on the proliferation, migration, and invasion of osteosarcoma cells has been unveiled; nevertheless, whether it also affects the stemness and epithelial-mesenchymal transition (EMT) of osteosarcoma cells is unclear. The purpose for this study was to explore the effects of CBR3-AS1 on the stemness and EMT of osteosarcoma cells as well as its underlying mechanism. qRT-PCR and western blot were applied to detect target gene expression. Function assays were conducted to evaluate the effect of genes on the stemness and EMT of osteosarcoma cells. Mechanism assays were done to verify the association among different genes. In vivo assays were also performed. The obtained data showed that CBR3-AS1 demonstrated a high expression in osteosarcoma cells. CBR3-AS1 could promote stemness and EMT of osteosarcoma cells as well as osteosarcoma tumor growth. Mechanically, CBR3-AS1 sponged miR-140-5p and recruited DDX54 to upregulate NUCKS1, thus activating the mTOR signaling pathway. Furthermore, NUCKS1 could facilitate stemness and EMT of osteosarcoma cells. In summary, this study reveals that CBR3-AS1 exerts an oncogenic role in osteosarcoma through modulating the network of the miR-140-5p/DDX54-NUCKS1-mTOR signaling pathway.

Retracted] MicroRNA-204 inhibits proliferation, migration, invasion and epithelial-mesenchymal transition in osteosarcoma cells via targeting Sirtuin 1

Following the publication of this paper, it was drawn to the Editors' attention by a concerned reader that certain of the cell Transwell assay data in the article (featured in Figs. 3B and 6B) were strikingly similar to data that appearing in different form in another article by different authors at different research institutions, which had already been published elsewhere at the time of the present article's submission. Owing to the fact that the contentious data in the above article had already appeared in different form in another article prior to its submission to Oncology Reports, the Editor has decided that this paper should be retracted from the Journal. The authors did not reply to indicate whether or not they agreed with the retraction of the paper. The Editor apologizes to the readership for any inconvenience caused. [the original article was published on Oncology Reports 34: 399‑406, 2015; DOI: 10.3892/or.2015.3986].

RETRACTED ARTICLE: MiR-151-3p transferred by cancer-associated fibroblast-derived extracellular vesicles promotes osteosarcoma progression through the CHL1/integrin 1\u03b2/TGF-\u03b2 axis

microRNA-151-3p (miR-151-3p) is a widely reported oncogene with a documented role in tumorigenesis and cancer progression. miRNAs are potent regulators of gene expression that are transferred between cells by extracellular vesicles (EVs). However, there is limited data about the role of EV-derived miR-151-3p in osteosarcoma (OS) progression, or the possible effects of miR-151-3p on the paracrine activity of cancer-associated fibroblasts (CAFs) in OS. To fill this gap of knowledge, we isolated CAF-derived EVs (CAF-EVs) from examined their up-take by human MG63 OS cells and their effects on MG63 biology. We found high expression of miR-151-3p in OS tissues, and miR-151-3p derived from CAF-EVs promoted the process of epithelial-mesenchymal transition (EMT), migration, and invasion of MG63 cells. Notably, bioinformatics analysis, RIP, and dual luciferase report test determined that CHL1 was the direct binding target of miR-151-3p. CAF-EVs mediated the CHL1/integrin 1β axis through miR-151-3p to regulate the TGF-β pathway and then promoted the proliferation, migration, invasion, and EMT of OS cells. Our in vivo results confirmed that EV secretion of miR-151-3p activated the TGF-β pathway through the CHL1/integrin 1β axis to promote the proliferation of OS cells and increase tumor volume and weight. Other effects were to upregulate E-Cadherin and downregulate the expression of N-Cadherin and β-catenin. Hence, our data reveal a potential functional axis mediated by EVs in the tumor microenvironment of OS and suggest potential candidate therapy targets.

LncRNA SNHG16 promotes epithelial-mesenchymal transition by upregulating ITGA6 through miR-488 inhibition in osteosarcoma

BackgroundOsteosarcoma is a primary cause of cancer-associated death in children and adolescents worldwide. Long non-coding RNAs SNHG16 (lncRNA SNHG16) and integrin subunit-a 6 (ITGA6) are recently reported to be involved in the tumorigenesis of osteosarcoma by multiple mechanisms. However, the correlation between SNHG16 and ITGA6 in osteosarcoma remains undetermined. MethodsExpression of miR-488, SNHG16 and ITGA6, as well as epithelial-mesenchymal transition (EMT) associated markers in osteosarcoma tissues and cell lines were examined by qRT-PCR or Western blotting. Effects of miR-488, SNHG16 and ITGA6 on cell migration, invasion were evaluated by wound-healing assay and transwell assay. Bioinformatics analysis and dual-luciferase reported assays were applied to assess the interaction among miR-488, SNHG16 and ITGA6. RNA immunoprecipitation (RIP) was also used to verify SNHG16 and miR-488 interaction. Finally, animal study was used to detect the effect of SNHG16 on osteosarcoma in vivo. ResultsSNHG16 and ITGA6 were significantly increased while miR-488 was decreased in osteosarcoma. ITGA6 was screened as a target gene of miR-488, and SNHG16 was sponged by miR-488 in osteosarcoma cells. MiR-488 overexpression and SNHG16 knockdown suppressed migration, invasion and EMT of osteosarcoma cells. Moreover, rescue assays proved that the influences of SNHG16 on osteosarcoma cells migration, invasion and EMT were dependent on miR-488 and ITGA6. In addition, the promotive effects of SNHG16 on osteosarcoma tumor growth and metastasis were further supported by xenograft tumor growth assay. ConclusionSNHG16 promoted migration, invasion and EMT of osteosarcoma by sponging miR-488 to release ITGA6.

Neuropilin and tolloid-like 2 regulates the progression of osteosarcoma.

Neuropilin and tolloid-like 2 (NETO2) is aberrantly expressed in various malignancies. However, its role in osteosarcoma (OS) remains to be elucidated. This study aimed to identify the function of NETO2 in OS cells. The expression of NETO2 in sarcoma tissues was determined using the GEPIA database, and the mRNA and protein expression of NETO2 in OS cells and OS tissue was also assessed. The biological effects of NETO2 on OS cells were determined by overexpressing and downregulating NETO2. Cell proliferation, invasion, migration, colony formation, and epithelial-mesenchymal transition in OS cells were evaluated. Consistent with the GEPIA database, expression of NETO2 was upregulated in human OS samples and cell lines. NETO2 overexpression not only promoted the proliferation, colony formation, invasion, and epithelial-mesenchymal transition of OS cells, but also activated the PI3K/AKT signaling. NETO2 downregulation resulted in opposite effects. Furthermore, after using an AKT inhibitor, the effects of NETO2 on OS cells were attenuated. In conclusion, this study showed that NETO2 functions as an oncogene of osteosarcomas by activating the PI3K/AKT pathway.

MiR-1224-5p Activates Autophagy, Cell Invasion and Inhibits Epithelial-to-Mesenchymal Transition in Osteosarcoma Cells by Directly Targeting PLK1 Through PI3K/AKT/mTOR Signaling Pathway.

BackgroundOsteosarcoma (OS) is one of the most common malignant bone tumors with a poor overall prognosis. MiR-1224-5p plays an important role in cancer, but its function and mechanism in OS have not been studied.Materials and MethodsThe expression of miR-1224-5p and PLK1 was detected by qRT-PCR in OS cells, adjacent tissues, and cell lines. Dual-luciferase reporter gene assay was used to verify the interaction between miR-1224-5p and PLK1. The expression of miR-1224-5p and PLK1 was intervened by transfection with miR-1224-5p mimic, NC mimic, pc-NC and PLK1, respectively. MTT, colony formation assay, Transwell and flow cytometry were used to observe the cell proliferation, invasion and apoptosis. Western blot was used to detect the expression levels of PLK1, PI3K/AKT/mTOR signaling pathway-related proteins, autophagy-related proteins, and epithelial-mesenchymal transition (EMT)-related proteins in the cells.ResultsWe found that miR-1224-5p was down-regulated and PLK1 expression was up-regulated in OS tissues and cells. On the other hand, it is further confirmed that PLK1 was a target gene of miR-1224-5p. Overexpression of miR-1224-5p inhibited the proliferation, invasion while promoted the apoptosis of OS cells, whereas overexpression of PLK1 promoted the proliferation, invasion and inhibited the apoptosis of OS cells. In the miR-1224-5p group (overexpression of miR-1224-5p), PI3K, AKT, and mTOR protein phosphorylation levels were significantly reduced, while autophagic activity was significantly activated, and the degree of EMT was significantly reduced. But the results in the PLK1 group (overexpression of PLK1) were the opposite. In addition, overexpression of miR-1224-5p reversed the effect of PLK1 upregulation on OS cells.ConclusionMiR-1224-5p targets PLK1 to inhibit PI3K/AKT/mTOR signaling pathway, thus mediating the proliferation, invasion, apoptosis, autophagy and EMT in OS cells.

The Long Noncoding RNA ZEB2-AS1 Contributes to Proliferation and Epithelial-to-Mesenchymal Transition of Osteosarcoma.

Background: Long non-coding RNA Zinc finger E-box binding homeobox 2 (ZEB2) antisense RNA 1 (ZEB2-AS1) has been shown to promote tumor progression. However, the clinical significance and fundamental function role of ZEB2-AS1 in osteosarcoma (OS) has been poorly understood. Methods: The expression of ZEB2-AS1 was determined in tumor tissues and matched normal tissues from 67 OS patients using quantitative reverse transcriptase PCR analysis. Clinical value of ZEB2-AS1 was evaluated by χ2 test and Kaplan-Meier method. Cell proliferation was analyzed using CCK-8 assay, colony formation. Cell apoptosis status was determined by caspase-3 activity assay. Cell migration, invasion and epithelial-mesenchymal transition (EMT) were investigated by scratch wound healing, transwell invasion assays and Western blotting. Results: Clinical association analysis revealed that high ZEB2-AS1 expression correlated with tumor size, distant metastasis and poor prognosis of OS patients. Moreover, ZEB2-AS1 expression was identified as an independent prognostic factor for OS patients. Loss-of-function assays demonstrated that ZEB2-AS1 knockdown suppressed the proliferation and induced apoptosis in OS cells. In addition, ZEB2-AS1 knockdown inhibited cell migration, invasion, EMT of OS cells in vitro. Conclusions: Taken together, our data demonstrate that ZEB2-AS1 serves a putative oncogenic role and associates with unfavorable prognosis in OS.

Berberine reverses epithelial-mesenchymal transition and modulates histone methylation in osteosarcoma cells.

Osteosarcoma is the most frequently occurring cancer in children as well as young adolescents and the metastatic forms worsen this condition to a further great extent. The metastatic dissemination of cancer cells is often acquired through a process of epithelial-mesenchymal transition (EMT). Since, phytochemicals have attracted intense interest in recent years due to their diverse pharmacological effects, in the present study, we investigated if berberine, a naturally occurring isoquinoline quaternary alkaloid, could modulate the EMT in osteosarcoma cells. Our experimental studies showed that berberine reduced cell viability, colony formation, wound healing ability and migration of osteosarcoma cells. Also, berberine significantly reduced the expression of matrix metalloproteinase (MMP)-2, suggesting its inhibitory action on the matrix metalloproteinases that are required for cancer cell invasion. The significant reduction in the expression of vimentin, N-cadherin, fibronectin and increased expression of E-cadherin further suggested its role in the inhibition of EMT in osteosarcoma cells. The downregulation of H3K27me3, as well as the decreased expression of the histone methyl transferase enzyme EZH2, further substantiated the fact that the plant alkaloid can be used as an epigenetic modulator in the treatment of osteosarcoma. In conclusion, our findings suggest that berberine inhibits proliferation and migration of osteosarcoma cells and most importantly reverses EMT along with modulation of key epigenetic regulators.

Circ_001569 regulates FLOT2 expression to promote the proliferation, migration, invasion and EMT of osteosarcoma cells through sponging miR-185-5p.

Osteosarcoma (OS) is a common malignant tumor in the world. Circular RNAs are endogenous non-coding RNAs that have been linked to the development of cancer. However, the role of circ_001569 in OS progression is still unclear. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of circ_001569, microRNA-185-5p (miR-185-5p) and flotillin-2 (FLOT2). The abilities of cell proliferation, migration and invasion were evaluated by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and Transwell assays, respectively. Also, western blot analysis was performed to assess the levels of epithelial–mesenchymal transition (EMT)-related proteins and FLOT2 protein. Besides, the dual-luciferase reporter assay was used to verify the interactions among circ_001569, miR-185-5p and FLOT2. Circ_001569 expression was increased in OS tissues and cells, and its knockdown reduced the proliferation, migration, invasion and EMT of OS cells. MiR-185-5p could interact with circ_001569. Inhibition of miR-185-5p could recover the suppression effects of silenced-circ_001569 on the proliferation and metastasis of OS cells. Furthermore, FLOT2 was a target of miR-185-5p. Overexpressed FLOT2 could restore the inhibition effects of miR-185-5p mimic on the proliferation and metastasis of OS cells. Also, FLOT2 expression was regulated by circ_001569 and miR-185-5p. In addition, circ_001569 knockdown also reduced the OS tumor growth in vivo. Circ_001569 might act as an oncogene in OS progression by regulating the miR-185-5p/FLOT2 axis, which provided a reliable new approach for the treatment of OS patients.

Downregulation of miR-22 Contributes to Epithelial-Mesenchymal Transition in Osteosarcoma by Targeting Twist1.

The epithelial-mesenchymal transition (EMT) is a vital step in osteosarcoma (OS) progression toward metastasis, but the specific molecular events governing this process are incompletely characterized, with miRNAs having increasingly been found to regulate the EMT. In this study, We assessed levels of miR-22 and its target, Twist1, via real-time PCR (qRT-PCR). We further used functional proliferation assays, measures of cell morphology, and western blotting to assess the functional relevance of miR-22 in OS and confirmed Twist1 as a miR-22 target via luciferase reporter assay. We observed a significant decrease in miR-22 levels in OS tumor samples relative to normal tissue, with such downregulating being significantly associated with tumor histological grade. When overexpressed, miR-22 impaired OS cell proliferation and EMT progression. We found Twist1 to be a direct miR-22 target, with levels of miR-22 and Twist1 mRNA being inversely correlated in patient samples. When overexpressed, miR-22 suppressed Twist1 translation and thereby attenuated the EMT in OS cells. These results clearly demonstrate that miR-22 can regulate the EMT in OS cells via targeting Twist1, thus highlighting a potentially novel pathway that can be therapeutically targeted in order to treat OS.

The Effects of Interleukin-33 (IL-33) on Osteosarcoma Cell Viability, Apoptosis, and Epithelial-Mesenchymal Transition are Mediated Through the PI3K/AKT Pathway.

BackgroundOsteosarcoma is the most common primary tumor of bone. Interleukin-33 (IL-33) is a pro-inflammatory cytokine that also participates in tumor progression. This study aimed to investigate the role of IL-33 in human osteosarcoma cell viability, proliferation, apoptosis, and epithelial-mesenchymal transition (EMT) in vitro and the molecular mechanisms involved.Material/MethodsThe normal osteoblast cell line, hFOB 1.19, and the human osteosarcoma cell lines SOSP-9607, SAOS2, MG63, and U2OS were studied. The expression of IL-33 mRNA and protein in human osteosarcoma cell lines were detected using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot, respectively. The effects of IL-33 on human osteosarcoma cell viability, apoptosis, EMT, and the signaling pathways were studied using the MTT assay, flow cytometry, qRT-PCR, and Western blot.ResultsIL-33 was upregulated in human osteosarcoma cell lines, including U2OS cells. The use of an IL-33 gene plasmid promoted osteosarcoma cell viability, inhibited cell apoptosis, increased the expression of Bcl-2, and reduced the expression of Bax. IL-33 reduced the level of E-cadherin and increased the levels of N-cadherin and matrix metalloproteinase-9 (MMP-9) in osteosarcoma cells at the mRNA and protein level. The use of the IL-33 plasmid increased the protein expression levels of p-AKT and the p-AKT/AKT ratio in osteosarcoma cells, and IL-33 siRNA reversed these findings.ConclusionsIL-33 was highly expressed in human osteosarcoma cells. Down-regulation of IL-33 reduced cell viability and EMT of osteosarcoma cells, and induced cell apoptosis through activation of the PI3K/AKT signaling pathway.

The Effects of MiR-214-3p and Irisin/FNDC5 on the Biological Behavior of Osteosarcoma Cells.

Background: Irisin/fibronectin type III domain-containing protein 5 (FNDC5) has important effects on breast cancer and liver cancer, however, its role in osteosarcoma is poorly understood. This study explored the effects of irisin/FNDC5 in osteosarcoma cells, aiming to provide a direction for treating osteosarcoma. Material and Methods: The expression levels of irisin/FNDC5 in serums and tissues of osteosarcoma patients and the expression characteristics of FNDC5 in osteosarcoma cell lines were measured. The effects of irisin, at different concentrations (0, 25, 50, 100, and 200 ng/mL), and FNDC5 on the viability, migration, and invasion of U2OS cells were analyzed. The target gene regulating FNDC5 was predicted, and its effects on irisin/FNDC5 and osteosarcoma cells were further explored. Results: The authors found that irisin/FNDC5 was significantly downregulated in the serums and tissues of osteosarcoma patients, and FNDC5 was also lowly expressed in osteosarcoma cell lines, especially in U2OS cells. Irisin/FNDC5 could not only inhibit the viability of U2OS cell in a concentration- and time-dependent manner but could also suppress cell migration and invasion. Furthermore, miR-214-3p inhibited the expression of irisin/FNDC5, and promoted the migration, invasion, and epithelial/mesenchymal transition (EMT) of U2OS cell through targeting FNDC5. Conclusions: Irisin/FNDC5 could inhibit the viability, migration, invasion, and EMT of osteosarcoma cells, and miR-214-3p could target FNDC5 to release its antitumor effects. Thus, irisin/FNDC5 and miR-214-3p might become a new direction for the treatment of osteosarcoma patients in the future.

MNX1-AS1 accelerates the epithelial-mesenchymal transition in osteosarcoma cells by activating MNX1 as a functional oncogene.

To investigate whether MNX1-AS1 can accelerate epithelial-mesenchymal transition (EMT) of osteosarcoma cells via activating MNX1. The expression pattern of MNX1-AS1 in osteosarcoma tissues and cell lines was examined by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Moreover, the cytoplasmic and nuclear levels of MNX1-AS1 in osteosarcoma cells were also determined. The regulatory effects of MNX1-AS1 on viability, clonality, migratory, and invasive abilities of the osteosarcoma cells were evaluated. The relative levels of MNX1 and EMT-related genes influenced by MNX1-AS1 were detected. The methylation ability in the promoter of the osteosarcoma cells transfected with si-MNX1-AS1 or MNX1-AS1 vector was determined by the whole genome bisulfite sequencing. MNX1-AS1 was upregulated in osteosarcoma tissues and cell lines, which was mainly expressed in the nucleus. The knockdown of MNX1-AS1 markedly attenuated viability, clonality, migratory, and the invasive abilities of the osteosarcoma cells. Besides, the transfection of si-MNX1-AS1 in U2OS and MG63 cells downregulated MNX1 and Snail, and upregulated E-cadherin. The methylation ability increased after the knockdown of MNX1-AS1, while the overexpression of MNX1-AS1 obtained the opposite trends. MNX1-AS1 mediates EMT of the osteosarcoma cells via activating MNX1, thereafter accelerating the progression of the osteosarcoma.

Diosgenin inhibits the epithelial-mesenchymal transition initiation in osteosarcoma cells via the p38MAPK signaling pathway.

Diosgenin is an important basic raw material for the production of steroid hormone drugs. It can be isolated and purified from a variety of traditional Chinese medicines or plants. Modern molecular biological studies have shown that diosgenin inhibits various tumor cells migration and invasion ability to varying degrees in vitro and in vivo. The aim of the present study was to observe the inhibitory effects of diosgenin on the invasive and metastatic capabilities of osteosarcoma cells and to determine the association between the effects of diosgenin on the epithelial-mesenchymal transition (EMT). Wound healing and Transwell assays were used to observe the inhibitory effects of diosgenin on the invasion and migration of two osteosarcoma cell lines. Immunofluorescence was used to observe changes in transforming growth factor β1 (TGF-β1) protein expression levels in the osteosarcoma cells following drug administration. EMT-associated proteins, including TGFβ1, E-cadherin and vimentin were detected by western blotting, which demonstrated that the drug may inhibit the initiation of EMT in osteosarcoma cells. Western blot analysis of the expression of all the proteins in the mitogen-activated protein kinase (MAPK) pathway demonstrated that the drug inhibited the MAPK signaling pathway. The primary mechanism of action of diosgenin was the inhibition of the phosphorylated p38 (pP38) protein. Through a combination of inhibitors of the p38MAPK signaling pathway and detection of the downstream EMT marker protein E-cadherin by quantitative PCR, pP38 was confirmed to be a target of diosgenin in the inhibition of EMT in the osteosarcoma cells via the MAPK molecular signaling pathway. Diosgenin may exhibit utility as an auxiliary drug for the clinical reduction of metastasis in patients with osteosarcoma.