Osteosarcoma Epithelial-mesenchymal Transition Research Articles

MFAP2 induces epithelial-mesenchymal transformation of osteosarcoma cells by activating the Notch1 pathway.

Osteosarcoma (OS) is a malignancy originating from mesenchymal tissue. Microfibril-associated protein 2 (MFAP2) plays a crucial role in cancer, notably promoting epithelial-mesenchymal transition (EMT). However, its involvement in OS remains unexplored. MFAP2 was silenced in U2OS cells using shRNA targeting MFAP2 (sh-MFAP2) and validated by quantitative real-time polymerase chain reaction (qRT-PCR). We extracted gene chip data of MFAP2 from multiple databases (GSE28424, GSE42572, and GSE126209). Correlation analyses between MFAP2 and the Notch1 pathway identified through the gene set variation analysis (GSVA) enrichment analysis were conducted using the Pearson correlation method. Cellular behaviors (viability, migration, and invasion) were assessed via the Cell Counting Kit-8 (CCK-8), wound healing, and Transwell assays. EMT markers (N-cadherin, vimentin, and β-catenin) and Notch1 levels were examined by western blotting and qRT-PCR. Cell morphology was observed microscopically to evaluate EMT. Finally, the role of MFAP2 in OS was validated through a xenograft tumor model. OS cell lines exhibited higher MFAP2 mRNA expression than normal osteoblasts. MFAP2 knockdown in U2OS cells significantly reduced viability, migration, and invasion, along with downregulation of N-cadherin and vimentin, as well as upregulation of β-catenin. MFAP2 significantly correlated with the Notch1 pathway in OS and its knockdown inhibited Notch1 protein expression. Furthermore, Notch1 activation reversed the inhibitory effects of MFAP2 knockdown on the malignant characteristic of U2OS cells. Additionally, MFAP2 knockdown inhibited tumor growth, expression levels of EMT markers, and Notch1 expression in OS tumor tissues. Our study revealed that MFAP2 was an upstream regulator of the Notch1 signaling pathway to promote EMT in OS. These findings suggested MFAP2 as a potential OS therapy target.

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.

Identification of an EMT-related gene-based prognostic signature in osteosarcoma.

The correlation between epithelial-mesenchymal transition (EMT) and osteosarcoma (OS) has been widely reported. Integration of the EMT-related genes to predict the prognosis is significant for investigating the mechanism of EMT in OS. Here, we aimed to construct a prognostic EMT-related gene signature for OS. Transcriptomic and survival data of OS patients were downloaded from Therapeutically Applicable Research to Generate Effective Treatments (TARGET) and Gene Expression Omnibus (GEO). We performed univariate Cox regression, least absolute shrinkage and selection operator (LASSO) regression, and stepwise multivariate Cox regression analysis to construct EMT-related gene signatures. Kaplan-Meier analysis and time-dependent receiver operating characteristic (ROC) were applied to evaluate its predictive performance. GSVA, ssGSEA, ESTIMATE, and scRNA-seq were performed to investigate the tumor microenvironment, and the correlation between IC50 of drugs and ERG score was investigated. Furthermore, Edu and transwell experiments were conducted to assess the malignancy of OS cells. We constructed a novel EMT-related gene signature (including CDK3, MYC, UHRF2, STC2, COL5A2, MMD, and EHMT2) for outcome prediction of OS. According to the signature, patients stratified into high- and low-ERG-score groups exhibited significantly different prognoses. ROC curves and Kaplan-Meier analysis revealed a promising performance of the signature with external validation. GSVA, ssGSEA, ESTIMATE algorithm, and scRNA-seq excavated EMT-related pathways and suggested the correlation between ERG score and immune activation. Notably, the pivotal gene CDK3 was upregulated in OS tissue and positively related to OS cell proliferation and migration. Our EMT-related gene signature might reference OS risk stratification and guide clinical strategies as an independent prognostic factor in OS.

Long Noncoding RNA PCGEM1 Facilitates Tumor Growth and Metastasis of Osteosarcoma by Sponging miR-433-3p and Targeting OMA1.

Osteosarcoma (OS) is regarded as one of the most common malignant bone tumors, mainly occurring in children and adolescents with high mortality. The dysregulation of lncRNAs is reported to regulate tumor development and be closely related to patient prognosis. Nevertheless, the role of long noncoding RNAs (lncRNAs) prostate-specific transcript 1 (PCGEM1) in OS remains uncharacterized. The current study aimed to explore the role of PCGEM1 in OS. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to examine the expression of PCGEM1 in OS cell lines. CCK-8, colony formation, Transwell, and western blotting analyses were applied to measure OS cell viability, proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) after PCGEM1 downregulation. Nuclear-cytoplasmic fractionation, RNA pulldown, RNA immunoprecipitation (RIP), luciferase reporter assays were performed to verify the relationship among PCGEM1, miR-433-3p. and OMA1 in OS. The xenograft tumor models were established to evaluate the effect of PCGEM1 on tumor growth of OS. In this study, we discovered that PCGEM1 knockdown inhibited cell proliferation, migration, invasion and EMT in OS (P < 0.05). Additionally, PCGEM1 directly bound to miR-433-3p (P < 0.01). OMA1 was confirmed to be a target gene of miR-433-3p (P < 0.05), positively regulated by PCGEM1 but negatively regulated by miR-433-3p. Rescue assays further verified that overexpression of OMA1 reversed the PCGEM1 knockdown-mediated inhibitory effect on the malignant phenotype in OS cells (P < 0.05). Moreover, knockdown of PCGEM1 inhibited tumor growth and metastasis in vivo (P < 0.05). Overall, PCGEM1 mediated tumor growth and metastasis of OS by sponging miR-433-3p and regulating OMA1, which might provide an innovative strategy for OS diagnosis or treatment.

RETRACTED ARTICLE: Histone methyltransferase SUV39H2 regulates LSD1-dependent CDH1 expression and promotes epithelial mesenchymal transition of osteosarcoma

ObjectiveOsteosarcoma (OS) is a malignant tumor characterized by the direct production of bone or osteoid tissues by proliferating tumor cells. Suppressor of variegation 3–9 homolog 2 (SUV39H2) is implicated in the occurrence of OS. Therefore, we designed this study to investigate effects of SUV39H2 in OS meditated by the lysine specific demethylase-1/E-cadherin (LSD1/CDH1) axis.MethodsClinical OS tissues and paracancerous tissues were collected for analysis of SUV39H2, LSD1 and CDH1 expression, and Kaplan–Meier survival analysis was applied to test the relationship between SUV39H2 expression and overall survival. Loss- and gain-of-function assays were conducted to determine the roles of SUV39H2, LSD1 and CDH1 in OS epithelial mesenchymal transition (EMT) and migration in OS cells, with quantitation of relevant proteins by immunofluorescence. We confirmed the effects of modulating the SUV39H2/CDH1 axis in a mouse OS tumor model.ResultsSUV39H2 and LSD1 were highly expressed, while CDH1 was downregulated in OS tissues and cells. SUV39H2 expression correlated inversely with overall survival of patients with OS. SUV39H2 positively regulated LSD1 expression, while LSD1 negatively regulated CDH1 expression. SUV39H2 or LSD1 overexpression, or CDH1 silencing promoted migration and EMT, as indicated by reduced E-cadherin and dramatically upregulated Vimentin and N-cadherin of OS cells. SUV39H2 expedited the progression of OS, which was reversed by CDH1 repression in the setting of OS in vitro and in vivo.ConclusionsCollectively, our results demonstrate highly expressed SUV39H2 in OS elevates the expression of LSD1 to downregulate CDH1 expression, thereby aggravating OS, providing a potential therapeutic target for treatment of OS.

Circular RNA Circ-03955 Promotes Epithelial-Mesenchymal Transition in Osteosarcoma by Regulating miR-3662/Metadherin Pathway.

Osteosarcoma is the most common primary malignant tumor, especially in children and adolescents. Circular RNAs (circRNAs) are found to play roles in the progression of osteosarcoma. However, the exact functions of circRNAs in osteosarcoma development still need to be clarified. We obtained differentially expressed circRNAs and miRNAs from a GSE99671 data set (GEO database). The gene co-expression network of ceRNAs and osteosarcoma-related genes was analyzed using the STRING database. qRT-PCR was used to detect the expression of circ-03955 and miR-3662. Transwell assays and flow cytometry were performed to detect phenotypic changes in cell function. A xenograft tumor model was established using BALB/c nude mice. Dual luciferase activity and RNA immunoprecipitation assays were performed to assess the relationship between circ-03955, miR-3662, and metadherin (MTDH). Immunohistochemistry, immunofluorescence, and Western blotting were used to assess protein expression levels. Circ-03955 was significantly upregulated, and miR-3662 was downregulated in osteosarcoma. Circ-03955 silencing inhibited the growth and metastasis of osteosarcoma. Mechanism analysis revealed that circ-03955 could bind to miR-3662, and the latter could target MTDH, leading to its suppressed expression and facilitating epithelial-mesenchymal transition (EMT). All these findings demonstrate that the presence of circ-03955 promotes EMT in osteosarcoma by acting as miR-3662 sponge-mediated MTDH expression.

MiR-196a promoted cell migration, invasion and the epithelial-mesenchymal transition by targeting HOXA5 in osteosarcoma.

Osteosarcoma (OS), aggressive neoplasms of the bone, is the most common primary bone cancer in children. MiR-196a usually low expressed in several tumors and its functions in osteosarcoma still unclear. Quantitative real-time polymerase chain reaction (qRT-PCR) was employed to assess the expression of miR-196a and the HOXA5. Cell metastasis and epithelial-mesenchymal transition (EMT) abilities were assessed using Transwell and western blot. The dual luciferase reporter assay was carried out to verify whether miR-196a directly targeted the 3'-untranslated region (UTR) of HOXA5 mRNA. MiR-196a was overexpressed and HOXA5 was low expressed in osteosarcoma versus the non-tumor tissues and normal cell lines. Upregulation of miR-196a or downregulation of HOXA5 was associated with worse outcome of osteosarcoma patients. MiR-196a enhanced cell migration, invasion and EMT by regulating the expression of HOXA5 through directly targeting the 3'-UTR of its mRNA in osteosarcoma. HOXA5 partially reversed roles of miR-196a on metastasis and EMT in osteosarcoma. MiR-196a promoted cell metastasis and EMT by targeting the 3'-UTR of HOXA5 mRNA in osteosarcoma. The newly identified miR-196a/HOXA5 axis provides novel insight into the pathogenesis of osteosarcoma.

MiR-363 suppresses cell migration, invasion, and epithelial-mesenchymal transition of osteosarcoma by binding to NOB1

BackgroundOsteosarcoma (OS) is a primary malignant bone tumor with a high rate of metastasis and a short 5-year survival rate. MiR-363 was downregulated in a variety of tumors and played a role in suppressing tumors. However, the roles of miR-363 in osteosarcoma remain unknown; thus, the purpose of this study was to explore the functions of miR-363 in osteosarcoma.MethodsCCK-8 and transwell assays were performed to evaluate the proliferation, migration, and invasion abilities of MG63 cells. The epithelial-mesenchymal transition (EMT) and apoptosis-associated proteins were measured by using Western blot assay. Luciferase reporter assay was utilized to verify whether miR-363 directly bound to the 3′-UTR of NOB1 mRNA.ResultsMiR-363 was downregulated while NOB1 was upregulated in osteosarcoma clinical tissue specimens and cell lines as compared with the adjacent normal tissue specimens and normal cell line. The miR-363 is reversely correlated with the expression of NOB1 in osteosarcoma tissues. Overexpression of miR-363 suppressed the ability of cell migration, invasion, and EMT, whereas low expression of miR-363 promoted this ability. In addition, miR-363 inhibited osteosarcoma proliferation both in vitro and in vivo and inhibited the apoptosis in MG63 cells. Interference of NOB1 could inhibit the migration, invasion, and EMT of osteosarcoma cell line MG63. NOB1 was verified to be a direct target of miR-363 and its expression was mediated by miR-363. Re-expression of NOB1 could partially reverse the inhibitory effect of miR-363 on cell migration and invasion. In addition, low expression of miR-363 or overexpression of NOB1 predicted poor prognosis of osteosarcoma patients.ConclusionMiR-363 inhibited osteosarcoma the proliferation, migration, invasion, and EMT and induced the apoptosis by directly targeting NOB1 in MG63 cells. The newly identified miR-363/NOB1 axis provides novel insights into the pathogenesis of osteosarcoma.

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.

CircSAMD4A regulates cell progression and epithelial‑mesenchymal transition by sponging miR‑342‑3p via the regulation of FZD7 expression in osteosarcoma

Osteosarcoma (OS) is a primary malignant tumor with a complex etiology. Therefore, research into the pathogenesis of osteosarcoma is considered a priority. Circular RNAs play important roles in cell metabolism and in the immune response and are closely associated with cancer treatment. However, research into the association of circular RNAs with osteosarcoma is limited. In the present study, CircSAMD4A was validated by RT-qPCR and agarose gel electrophoresis. CircSAMD4A and miR-342-3p expression was detected by RT-qPCR. The relative protein expression levels were measured by western blot analysis. MTT assay and flow cytometry were used to detect cell cytotoxicity and apoptosis, respectively. Transwell assay was applied to assess cell migration and invasion. Dual-luciferase reporter assay was used to determine the association among CircSAMD4A, Frizzled-7 (FZD7) and miR-342-3p. In vivo, subcutaneous tumor formation assay was performed in an experiment with nude mice. The results revealed that the expression levels of CircSAMD4A and FZD7 were upregulated, while those of miR-342-3p were downregulated in OS tissues and cells. The inhibition of CircSAMD4A suppressed cell progression and epithelial-mesenchymal transition (EMT), and promoted cell apoptosis in OS. The reduction of miR-342-3p reversed the effects of CircSAMD4A downregulation on cell cytotoxicity, migration, invasion, apoptosis and EMT in OS, while FZD7 overexpression blocked the effect of miR-342-3p upregulation on OS progression. The suppressive effect of sh-CircSAMD4A on tumor growth was thus verified in OS. Overall, the present study demonstrated that CircSAMD4A affected cell cytotoxicity, invasion, apoptosis, migration and EMT by regulating the miR-342-3p/FDZ7 axis in OS, thereby providing a novel regulatory mechanism and a potential therapeutic target for OS.

Notch Signaling Pathway Regulates Epithelial-mesenchymal Transition of Osteosarcoma by Activating Slug

Notch Signaling Pathway Regulates Epithelial-mesenchymal Transition of Osteosarcoma by Activating Slug

Tumor-associated macrophages promote lung metastasis and induce epithelial-mesenchymal transition in osteosarcoma by activating the COX-2/STAT3 axis

Osteosarcoma (OS) is a common, malignant musculoskeletal tumor in young people. Neoadjuvant chemotherapy has improved the survival of osteosarcoma patients but with limited benefit due to metastasis. Tumor-associated macrophages (TAMs) are involved in various mechanisms of tumor biology, which include oncogenesis, drug resistance, and tumor immune escape, as well as tumor metastasis. In this study, we proved that TAMs possess the ability to promote OS cell migration and invasion by upregulating COX-2, MMP9, and phosphorylated STAT3 and to induce the epithelial-mesenchymal transition (EMT). This evidence has also been verified in a tumor-bearing animal model, and in OS patients. Furthermore, we observed the anti-metastasis effect of COX-2 inhibition by repressing COX-2 expression, EMT-activating transcription factors and the STAT3 pathway, both in vitro and in vivo. We propose that TAMs promote OS metastasis and invasion by activating the COX-2/STAT3 axis and EMT. These findings suggest that TAMs and COX-2 may be potential targets for future anti-metastasis therapy.

Matrix stiffness regulates epithelial-mesenchymal transition via cytoskeletal remodeling and MRTF-A translocation in osteosarcoma cells

Matrix stiffness is known to alter cellular behaviors in various biological contexts. Previous investigations have shown that epithelial–mesenchymal transition (EMT) promotes the progression and invasion of tumor. Mechanical signaling is identified as a regulator of EMT. However, the molecular mechanisms underlying the influence exerted by matrix stiffness on EMT in osteosarcoma remains largely unknown. Using polyacrylamide hydrogel model, we investigate the effects of matrix stiffness on EMT and migration in osteosarcoma. Our data indicates that high matrix stiffness regulates cell morphology and promotes EMT and migration in osteosarcoma MG63 cell line in vitro. Notably, matrix stiffness promotes polymerization of actin and nuclear accumulation of myocardin-related transcription factor A (MRTF-A). Furthermore, inhibiting MRTF-A by CCG 203971 significantly reduces EMT and migration on rigid gels. These data suggest that matrix stiffness of the tumor microenvironment actively regulate osteosarcoma EMT and migration through cytoskeletal remodeling and translocation of MRTF-A, which may contribute to cancer progression.

MiR-17-5p promotes proliferation and epithelial-mesenchymal transition in human osteosarcoma cells by targeting SRC kinase signaling inhibitor 1.

MicroRNA-17-5p (miR-17-5p) and epithelial-mesenchymal transition (EMT) have been reported to participate in the development and progression of multiple cancers. However, the relationship between the miR-17-5p and EMT in osteosarcoma (OS) is still poorly understood. This study was to investigate the effects of the miR-17-5p and its potential mechanism in regulating proliferation, apoptosis, and EMT of human OS. Quantitative real-time PCR was used to detect the miR-17-5p and SRC kinase signaling inhibitor 1 (SRCIN1) messenger RNA expression in OS specimens and cell lines. After transfection with miR-17-5p inhibitors, proliferation, apoptosis, migration, and invasion of OS cells were assessed by using the Cell Counting Kit-8, the annexin V-FITC apoptosis, wound-healing, and transwell assays. The SRCIN1 was validated as a target of the miR-17-5p through bioinformatics algorithms and luciferase reporter assay. Moreover, the expression of EMT markers, E-cadherin, N-cadherin, and Snail was identified by the Western blot analysis. MiR-17-5p was significantly upregulated in OS tumor samples and cell lines. It inhibited proliferation and EMT, and promoted apoptosis in OS. The SRCIN1 was identified as a direct target of the miR-17-5p. Silenced miR-17-5p could change the expression of EMT markers, such as upregulating the expression of E-cadherin, and downregulating the expression of N-cadherin and Snail through targeting the antioncogenic SRCIN1. These findings suggest that the miR-17-5p promotes cell proliferation, and EMT in human OS by directly targeting the SRCIN1, and reveal a branch of the miR-17-5p/SRCIN1/EMT signaling pathway involved in the progression of OS.

Disruption of IGF‑1R signaling by a novel quinazoline derivative, HMJ‑30, inhibits invasiveness and reverses epithelial-mesenchymal transition in osteosarcoma U‑2 OS cells.

Osteosarcoma is the most common primary malignancy of the bone and is characterized by local invasion and distant metastasis. Over the past 20 years, long-term outcomes have reached a plateau even with aggressive therapy. Overexpression of insulin-like growth factor 1 receptor (IGF-1R) is associated with tumor proliferation, invasion and migration in osteosarcoma. In the present study, our group developed a novel quinazoline derivative, 6-fluoro-2-(3-fluorophenyl)-4-(cyanoanilino)quinazoline (HMJ-30), in order to disrupt IGF-1R signaling and tumor invasiveness in osteosarcoma U-2 OS cells. Molecular modeling, immune-precipitation, western blotting and phosphorylated protein kinase sandwich ELISA assays were used to confirm this hypothesis. The results demonstrated that HMJ-30 selectively targeted the ATP-binding site of IGF-1R and inhibited its downstream phosphoinositide 3-kinase/protein kinase B, Ras/mitogen-activated protein kinase, and IκK/nuclear factor-κB signaling pathways in U-2 OS cells. HMJ-30 inhibited U-2 OS cell invasion and migration and downregulated protein levels and activities of matrix metalloproteinase (MMP)-2 and MMP-9. An increase in protein levels of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 was also observed. Furthermore, HMJ-30 caused U-2 OS cells to aggregate and form tight clusters, and these cells were flattened, less elongated and displayed cobblestone-like shapes. There was an increase in epithelial markers and a decrease in mesenchymal markers, indicating that the cells underwent the reverse epithelial-mesenchymal transition (EMT) process. Overall, these results demonstrated the potential molecular mechanisms underlying the effects of HMJ-30 on invasiveness and EMT in U-2 OS cells, suggesting that this compound deserves further investigation as a potential anti-osteosarcoma drug.

LncRNA, CRNDE promotes osteosarcoma cell proliferation, invasion and migration by regulating Notch1 signaling and epithelial-mesenchymal transition

Osteosarcoma is the most common bone malignancy in adolescence. Recently, the long non-coding RNAs (lncRNAs) were reported to play important roles in osteosarcoma progression. The present study examined the potential role of the lncRNA, Colorectal Neoplasia Differentially Expressed (CRNDE) and molecular mechanisms underlying osteosarcoma progression. In the present study, we identified that CRNDE was up-regulated in osteosarcoma tissues and cell lines, and CRNDE expression level was significantly higher in osteosarcoma tissues from patients with advanced stage and metastasis. Overexpression of CRNDE promoted cell growth, cell proliferation, cell invasion and migration, and increased cell population at S phase with a decreased cell population at G0/G1 phase in MG-63 cells. Knock-down of CRNDE suppressed cell growth, cell proliferation, cell invasion and migration, and decreased cell population at S phase with an increased cell population at G0/G1 phase in U2OS cells. Overexpression of CRNDE was found to enhance the activity of Notch1 signaling and promote epithelial-mesenchymal transition (EMT) in MG-63 cells, while knock-down of CRNDE exerted the opposite effects in U2OS cells. The in vivo results showed that knock-down of CRNDE suppressed the tumor growth in the nude mice inoculated with osteosarcoma cells, and knock-down of CRNDE also suppressed the mRNA expression of Notch1, JAG1, N-cadherin, vimentin, and increased the mRNA expression of E-cadherin in the tumor tissues. Collectively, our results indicated that CRNDE functioned as an oncogene in osteosarcoma cell lines, and CRNDE may exert its oncogenic role via regulating Notch1 signaling and EMT in osteosarcoma.

WITHDRAWN: LncRNA NEAT1 regulates epithelial-mesenchymal transition of osteosarcoma by miR-186-5p as competing endogenous RNA

// Hongyu Tan 1 and Liang Zhao 1 1 Department of Orthopedics, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan, China Correspondence to: Hongyu Tan, email: hongyu7780@163.com Keywords: osteosarcoma; lncRNA NEAT1; miR-186-5p; HIF-1α; ceRNA Received: August 23, 2017 Accepted: October 28, 2017 Published: November 30, 2017 ABSTRACT Increasing evidence showed that lncRNA NEAT1 play important roles in regulating tumor carcinogenesis and progression. However, the function and underlying mechanism of NEAT1 in osteosarcoma (OS) remain unclear. In the present study, we found that NEAT1 expression was significantly upregulated in OS tissues and cell lines. High NEAT1 expression was closely associated with advanced clinicopathologic features and poor overall survival of OS patients. In vitro function assay, we found that NEAT1 could promote OS cells proliferation, invasion, and epithelial-mesenchymal transition (EMT) process. NEAT1 could also promote OS cells growth in vivo . In addition, our studies showed that miR-186-5p was a downstream target of NEAT1 in OS. Functionally, miR-186-5p suppressed OS cells proliferation, invasion, and EMT process. Furthermore, our data revealed that HIF-1α was a downstream target of miR-186-5p and NEAT1 could exert its tumor oncogenic roles on OS cells via miR-186-5p/HIF-1α axis. Taken together, we elucidated that the NEAT1/miR-186-5p/HIF-1α axis might be a therapeutic approach for the treatment of OS.

Long Noncoding RNA GAS5 Suppresses Cell Growth and Epithelial-Mesenchymal Transition in Osteosarcoma by Regulating the miR-221/ARHI Pathway.

Dysregulated long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) play key roles in the development of human cancers. The lncRNA growth arrest-specific 5 (GAS5) is reported to be a tumor suppressor in multiple cancers. However, the roles of GAS5 and its related miRNAs in osteosarcoma are poorly understood. This study explored the potential functions and mechanisms of GAS5 in the tumorigenesis of osteosarcoma. Here, the expression of GAS5, miR-221 and aplasia Ras homologue member I (ARHI) was determined in osteosarcoma tissues and cells by Real-time PCR (RT-qPCR). The underlying mechanism of GAS5 in osteosarcoma growth was analyzed via MTT, Transwell, RT-qPCR, Western blot, dual-luciferase reporter assay, RNA immunoprecipitation, and xenograft models after GAS5 overexpression. GAS5 and ARHI levels were significantly reduced, while miR-221 increased, both in osteosarcoma tissues and cells. Overexpression of GAS5 suppressed the proliferation, migration, and epithelial-mesenchymal transition (EMT) of osteosarcoma cells. GAS5 could directly bind to miR-221 to decrease miR-221 expression and enhance ARHI expression. The effect of GAS5 overexpression on the proliferation, migration and EMT was reversed by miR-221 mimics or ARHI siRNA in osteosarcoma cells. Additionally, GAS5 suppressed tumor volume, Ki-67 and PCNA staining, and EMT process in the development of osteosarcoma in vivo. Taken together, lncRNA GAS5 functions as a competing endogenous RNA for miR-221 to suppress cell growth and EMT in osteosarcoma by regulating the miR-221/ARHI pathway. J. Cell. Biochem. 118: 4772-4781, 2017. © 2017 Wiley Periodicals, Inc.

Sex-determining region Y-box protein 3 induces epithelial-mesenchymal transition in osteosarcoma cells via transcriptional activation of Snail1

BackgroundThe transcription factor sex-determining region Y-box protein 3 (SOX3) plays important roles in various types of cancer. However, its expression and function have not yet been elucidated in osteosarcoma (OS).MethodsThe expression levels of SOX3 in OS tissues and OS cell lines were determined by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis. The effects of SOX3 expression on OS cell biological traits were investigated by overexpressing and downregulating SOX3 protein. The expression of epithelial-mesenchymal transition (EMT) markers and transcription factors associated with EMT (EMT-TFs), were detected simultaneously. The mechanism underlying SOX3-mediated Snail1 expression was further investigated.ResultsSOX3 was upregulated in human OS tissues. SOX3 overexpression promoted the EMT, migration and invasion in OS cells. The downregulation of SOX3 resulted in opposing effects. Furthermore, SOX3 upregulation enhanced the expression of the transcriptional repressor Snail1 by binding to its promoter region. Additionally, a positive correlation among the expression of SOX3, Snail1, and E-cadherin was demonstrated in human OS tissues.ConclusionsSOX3 promotes migration, invasiveness, and EMT in OS cells via transcriptional activation of Snail1 expression, suggesting that SOX3 is a novel regulator of EMT in OS and may serve as a therapeutic target for the treatment of OS metastasis.

High expression of TRIM29(ATDC) contributes to poor prognosis and tumor metastasis by inducing epithelial‑mesenchymal transition in osteosarcoma.

The association of TRIM29 overexpression with cancer progression and poor clinical prognosis has been reported in the context of several types of cancers. In the present study, we investigated the prognostic relevance of TRIM29 and its involvement in the progression of human osteosarcoma. To the best of our knowledge, this is the first study to demonstrate a major role of TRIM29 in osteosarcoma. Our results showed that the expression of TRIM29 in osteosarcoma tissues was much higher than that in normal bone tissues. Furthermore, TRIM29 expression was significantly correlated with tumor size, recurrence, metastasis and overall survival time. High expression of TRIM29 and presence of metastasis were independent predictors of poor prognosis in thesepatients. Both protein and mRNA expression of TRIM29 in osteosarcoma cell lines were significantly higher than those in osteoblast cell line, hFOB1.19. Moreover, the results indicated that TRIM29 promoted migration and invasive growth of osteosarcoma cells by inducing epithelial-mesenchymal transition. Therefore, ectopic expression of TRIM29 potentially contributes to metastasis and poor prognosis inpatients with osteosarcoma. In summary, TRIM29 is a potential prognostic biomarker and a therapeutic target forpatients with osteosarcoma.