RNF130 inhibits the proliferation, migration and invasion of osteosarcoma through DAB1 mediated suppression of the PI3K/AKT signaling pathway.

The anti-tumor action of engineered mesenchymal stem cells highlights the double-edged role of oncoproteins in osteosarcoma, and suggests the possibility of developing a novel strategy for protein-based cancer therapy.

Forkhead box N3 (FOXN3) has been reported to be downregulated in numerous cancers, including laryngeal, oral squamous cell and hepatocellular carcinomas, and diffuse large B‑cell lymphoma. FOXN3 was proposed to serve as a tumor suppressor; however, the function of FOXN3 in osteosarcoma (OS) remains unknown. The present study suggested that FOXN3 was notably downregulated in OS tissues compared with in adjacent normal tissues, and the expression of FOXN3 was negatively correlated with tumor size, metastasis and tumor, node and metastasis stage. Additionally, low expression levels of FOXN3 predicted a poor prognosis of patients with OS. Additionally, the present study revealed that FOXN3 was also downregulated in OS cells. Numerous functional experiments, including colony formation, Cell Counting Kit‑8, wound healing and Transwell invasion assays, were performed. The results of the present study revealed that FOXN3 suppressed the proliferation, migration and invasion of OS cells. SIRT6 has been reported to serve a key role in OS; chromatin‑immunoprecipitation (ChIP) and quantitative ChIP, as well as a luciferase reporter assay, demonstrated that SIRT6 was transcriptionally regulated by FOXN3. Furthermore, FOXN3 also regulated matrix metalloproteinase‑9 secretion via the regulation of SIRT6 expression. The findings of the present study indicated that FOXN3 serves as a tumor suppressor in OS and proposed FOXN3 as a prognostic predictor and a therapeutic target for patients with OS.

Osteosarcoma (OS) is the most common bone cancer worldwide. There is evidence that microRNA-409 (miR-409-3p) is involved in tumorigenesis and cancer progression, however, its possible role in OS requires clarification. In the present study, we evaluated the expression level, clinical significance, and mode of action of miR-409-3p in OS. The miR-409-3p levels were diminished in the OS cells and tissues compared with associated adjacent non-tumor tissues and a non-cancer osteoplastic cell line. Low miR-409-3p expression levels were associated with clinical stage and distant metastasis in patients with OS. Resumption of miR-409-3p expression attenuated OS cell proliferation and invasion. Additionally, based on informatics analyses, we predicted that zinc-finger E-box-binding homeobox-1 (ZEB1) is a possible target of miR-409-3p. This hypothesis was confirmed using luciferase reporter assays, reverse transcription-quantitative real-time polymerase chain reaction, and Western blot analyses. The findings of the current study indicated that ZEB1 was up-regulated in the OS tissues and cell lines, and that this up-regulation was inversely proportional to miR-409-3p expression levels. Furthermore, down-regulation of ZEB1 decreased OS cell invasion and proliferation, illustrating that the tumor suppressive role of miR-409-3p in OS cells may be exerted via negative regulation of ZEB1. Taken together, our observations highlight the potential role of miR-409-3p as a tumor suppressor in OS partially through down-regulation of ZEB1 and suggest that miR-409-3p has potential applications in OS treatment.

We aim to investigate the effect of miR-106a-5p on the proliferation, migration, and invasion of osteosarcoma (OS) cells by targeting high-mobility group AT-hook 2 (HMGA2). Real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) was used for detecting the expressions of miR-106-5p and HMGA2 in 137 OS and adjacent normal bone tissues. Immunohistochemistry was applied for the HMGA2 protein expression detection. Luciferase reporter gene assay was conducted for verifying whether miR-106-5p targeted HMGA2. MG63 and U2SO cells were respectively divided into five groups: Blank, miR-106a-5p, scramble, HMGA2-siRNA, and miR-106a-5p+HMGA2 groups. RT-qPCR and western blot were applied for detecting the expressions of miR-106a-5p and HMGA2 in five groups. Proliferation rate, cell cycle, invasion, and migration ability of OS cells were detected using methyl thiazolyl-tetrazolium, 5-ethynyl-2'-deoxyuridine (Edu) assay, flow cytometry, and Transwell. Compared with adjacent normal tissues, OS tissues presented with decreased miR-106a-5p expressions, elevated HMGA2 mRNA, and positive expressions (all p < 0.05). The sensitivity and specificity of miR-106a-5p were 97.8%, 93.43%, and HMGA2 mRNA were 97.8%, 99.27%, separately. miR-106a-5p and HMGA2 expressions were associated with tumor size, Enneking stage, distant metastasis, and lung metastasis. Expressions of HMGA2 in OS cells in miR-106a-5p and HMGA2 siRNA groups were both significantly decreased with the same downregulation level, and the proliferation rates in both groups were obviously slowed down after 48 h (both p < 0.001). Edu positive cells, S phase cells (majority of cells blocked at G0/G1 phase), migratory and invasive cells were obviously decreased (all p < 0.05). Downregulation of miR-106a-5p was found in OS tissues, and upregulation of miR-106a-5p can inhibit the proliferation, migration, and invasion by targeting HMGA2 in OS cells.

The aim of the present study was to clarify the effect of long non-coding RNA (lncRNA) small nucleolar RNA host gene 1 (SNHG1) on the proliferation, migration and invasion of osteosarcoma (OS) cells and to explore the potential underlying mechanisms. The expression levels of SNHG1, microRNA (miR)-424-5p and fibroblast growth factor 2 (FGF2) in OS tissues and cells were detected using reverse transcription-quantitative polymerase chain reaction. OS cell proliferation, migration and invasion were analysed by MTT, wound healing and Transwell invasion assays, respectively. The targeting relationships between SNHG1 and miR-424-5p, as well as between miR-424-5p and FGF2, were confirmed using RNA-binding protein immunoprecipitation and/or dual-luciferase reporter gene assays. The results demonstrated that the expression levels of SNHG1 and FGF2 were upregulated, whereas the expression of miR-424-5p was downregulated in OS tissues and cells. The silencing of SNHG1 significantly inhibited the proliferation, migration and invasion of OS cells. Additionally, FGF2 was shown to be a target of miR-424-5p, which in turn, was a target of SNHG1. miR-424-5p silencing and FGF2 overexpression both reversed the suppressive effects of SNHG1 knockdown on the proliferation, migration and invasion of OS cells. Thus, the silencing of SNHG1 may inhibit the proliferation, migration and invasion of OS cells by regulating the miR-424-5p/FGF2 axis.

Role of let-7 family in the invasion and metastasis of osteosarcoma.

MicroRNAs (miRs) are a class of small non-coding RNAs and have key roles in various cancer types. Recently, miR-503 has been reported to act as a tumor suppressor in osteosarcoma. However, the detailed mechanism of the regulatory role of miR-503 in osteosarcoma cell proliferation and invasion has largely remained elusive. The present study found that miR-503 was significantly downregulated in osteosarcoma tissues compared to that in matched adjacent non-tumorous tissues. In addition, the expression of miR-503 in osteosarcoma of T3-T4 stage was significantly lower when compared with that in T1-T2 stage samples. miR-503 was also downregulated in osteosarcoma cell lines (Saos-2, MG63, U2OS and SW1353), when compared with that in the normal osteoblast cell line hFOB. Overexpression of miR-503 significantly inhibited the proliferation and invasion of U2OS cells and decreased the protein levels of insulin-like growth factor 1 receptor (IGF-1R), which was further identified as a novel target of miR-503 by a luciferase reporter assay. Moreover, overexpression of IGF-1R eliminated the suppressive effects of miR-503 on the proliferation and invasion of U2OS cells, suggesting that miR-503 inhibits osteosarcoma cell proliferation and invasion by directly targeting IGF-1R. Furthermore, IGF-1R was significantly upregulated in osteosarcoma tissues compared with that in adjacent non-tumor tissues, as well as in osteosarcoma cell lines compared with that in hFOB cells. In addition, the expression levels of IGF-1R were inversely correlated to the miR-503 levels in osteosarcoma tissues, suggesting that the increased IGF-1R expression may be caused by the reduced expression of miR-503. In conclusion, the present study demonstrated that miR-503 suppresses cell proliferation and invasion in osteosarcoma via targeting IGF-1R and thus highlights the importance of miR-503/IGF-1R signaling in the malignant progression of osteosarcoma.

MicroRNAs (miRNAs or miRs) are involved in the development and progression of numerous types of cancer however their role in osteosarcoma has not been fully clarified. The present study aimed to use high-throughput bioinformatics analysis as well as in vitro experiments to investigate the potential role of transcription factors, miRNAs and their targets in the progression of osteosarcoma. miRNA data and clinical information of osteosarcoma were obtained from Gene Expression Omnibus database to investigate differentially expressed miRNAs. The expression of miRNAs/mRNAs in osteosarcoma cell lines was detected via reverse transcription-quantitative (RT-qPCR). MTT and colony formation assay were used to determine cell proliferation ability and transwell assay was used to observe cell invasion and migration ability. A total of four prediction algorithms for miRNA-mRNA interactions were used to determine potential target genes of miR-487a. Predicted target genes were used to intersect with overlapped differentially expressed genes (DEGs) from GSE12865 and The Cancer Genome Atlas osteosarcoma datasets. Expression of NK3 homeobox 1 (NKX3-1) was analyzed by western blotting and RT-qPCR assay. Dual luciferase assay was conducted to verify whether NKX3-1 was a direct target of miR-487a. The regulatory association between Kruppel-like factor 5 (KLF5) and miR-487a was detected using chromatin immunoprecipitation assay. miR-487a was upregulated in osteosarcoma tissue (GSE65071 and GSE28423) and cell lines (HOS and MG63). miR-487a mimic promoted proliferation, migration and invasion of osteosarcoma cells. NKX3-1 was a direct target of miR-487a and transfection of NKX3-1 plasmid reversed the effect of miR-487a on proliferation, migration and invasion of osteosarcoma cells. KLF5 enhanced miR-487a expression by directly binding to its promoter region and miR-487a inhibitor reversed the effect of KLF5 on proliferation, migration and invasion of osteosarcoma cells. The present results indicated that KLF5/miR-487a signaling promoted invasion and metastasis of osteosarcoma cells via targeting NKX3-1.

Down-regulation of microRNA-31-5p inhibits proliferation and invasion of osteosarcoma cells through Wnt/β-catenin signaling pathway by enhancing AXIN1

Osteosarcoma is the most common primary malignant bone tumor, which occurs in adolescents. As reported by our previous studies, HER4 indicates a poor prognosis of primary osteosarcoma. However, its mechanisms in the pathogenesis of osteosarcoma have not yet been studied. The purpose of this study was to investigate the role of HER4 in osteosarcoma and whether the PI3K/AKT pathway is involved. In this study, western blot analysis was used to investigate the expression of HER4 protein in osteosarcoma tissues and cell lines. CCK8 and transwell assays were used to detect the effects of HER4 on the proliferation, migration, and invasion of osteosarcoma cells in vitro. The effects of HER4 on the growth and metastasis of osteosarcoma in vivo were detected by tumor formation and immunofluorescence in nude mice. The role of the PI3K/AKT pathway in HER4 regulation of the growth and metastasis of osteosarcoma was examined by western blot analysis and immunofluorescence assay. We found that HER4 protein was highly expressed in clinical osteosarcoma specimens and osteosarcoma cells. HER4 markedly promoted the proliferation, migration, and invasion of osteosarcoma cells in vitro as well as the growth and metastasis of osteosarcoma in vivo. HER4 overexpression upregulated the expression of phosphorylated protein kinase B (pAKT), proliferation marker antigen Ki67, and metastasis cell marker matrix metalloproteinase 9 (MMP9). Notably, PI3K/AKT inhibitor LY294002 significantly inhibited the effects of HER4 via the downregulation of pAKT, Ki67, and MMP9. Moreover, LY294002 markedly blocked the effects of HER4-induced upregulation of tumor malignancy. The present study suggests that HER4 may promote the growth and metastasis of osteosarcoma via the PI3K/AKT pathway. The HER4/PI3K/AKT pathway could serve as a potential target for the treatment of osteosarcoma.

BackgroundMicroRNAs are known to regulate carcinogenesis of osteosarcoma. Although, miR-16-5p is known to exert inhibitory effects on several forms of cancers, its effects on the growth and invasion of osteosarcoma have not been studied.MethodsWe collected human osteosarcoma specimens and adjacent tissues to detect the expression of miR-16-5p by real-time polymerase chain reaction, immunoblotting, and immunohistochemistry. The proliferation, migration, and invasion of MG63 and HOS cells following miR-16-5p overexpression and inhibition were detected with cell counting kit-8, wound healing assay, and Transwell assay, respectively. An expression vector carrying a mutated 3′-untranslated region of mothers against decapentaplegic homolog 3 (Smad3) was constructed.ResultsThe results showed that miR-16-5p expression was downregulated in osteosarcoma tissues and cells as compared with adjacent counterparts, while Smad3 was overexpressed in osteosarcoma cells. The overexpression of miR-16-5p resulted in the inhibition of the proliferation, migration, and invasion of osteosarcoma cells and enhanced the therapeutic effect of cisplatin. These effects were attenuated with miR-16-5p expression inhibition. In cells transfected with miR-16-5p mimic, Smad3 expression decreased, while this effect was absent in the cells carrying mutated Smad3.ConclusionsTherefore, miR-16-5p inhibits the growth and invasion of osteosarcoma by targeting Smad3.

BackgroundLong non-coding RNA X-inactive specific transcript (XIST) regulates the progression of a variety of tumors, including osteosarcoma. Bone marrow mesenchymal stem cells (BMSCs) can be recruited into osteosarcoma tissue and affect the progression by secreting exosomes. However, whether BMSCs derived exosomes transmit XIST to regulate the growth and metastasis of osteosarcoma and the related mechanism are still unclear.MethodIn this study, BMSCs derived exosomes were used to treat human osteosarcoma cells MG63 and 143B, and the level of XIST in BMSCs was intervened by siRNA. CCK-8, EdU, transwell assays were used to analyze the changes of cell proliferation, migration and invasion. Bioinformatics analysis, RNA pulldown and dual-luciferase reporter gene assays validated the targeted relationship of XIST with miR-655 and the interaction between miR-655 and ACLY 3’-UTR. 143B/LUC cell line was used to establish an animal model of in situ osteosarcoma to verify the found effects of XIST on osteosarcoma. Oil Red O staining, Western blot and so on were used to detect the changes of lipid deposition and protein expression.ResultsIt was found that BMSCs derived exosomes promoted the proliferation, migration and invasion of osteosarcoma cells, and the down-regulation of XIST inhibited this effect. miR-655 mediated the role of BMSCs derived exosomal XIST in promoting the progression of osteosarcoma and down-regulation of miR-655 could reverse the effects of inhibiting XIST on the proliferation, migration and invasion of osteosarcoma cells. Meanwhile, animal level results confirmed that BMSCs derived exosomal XIST could promote osteosarcoma growth and lung metastasis by combining with miR-655. In-depth mechanism study showed that BMSCs derived exosomal XIST combined with miR-655 to increase the protein level of ACLY, which led to lipid deposition and activate β-catenin signal to promote the proliferation, migration and invasion of osteosarcoma cells.ConclusionThis study showed that BMSCs derived exosomal XIST could enter osteosarcoma cells, bind and down-regulates the level of miR-655, resulting in an increase in the level of ACLY, thus increasing the lipid deposition and the activity of β-catenin signal to promote the growth and metastasis of osteosarcoma.

Background: Osteosarcoma is the most common primary bone malignancy in children and adolescents, and the pathogenesis of this cancer remains unclear. Therefore, the discovery of new biomarkers for the diagnosis, prognosis, and treatment of osteosarcoma remains an important but unmet clinical need. Method: Quantitative real-time PCR was carried out to examine the expression of miR-23a. Methylation-specific PCR was performed to evaluate the DNA methylation status of the miR-23a promoter. Cell proliferation, migration, and invasion were examined by cell counting assays, wound healing assays, and cell invasion assays, respectively. Western blot analysis and luciferase reporter assays were performed to identify miR-23 target genes. Nude mice were used to investigate the function of miR-23a in vivo. Results: The expression of miR-23a was decreased in osteosarcoma cells and tissues compared to normal controls. The promoter region of the miR-23a gene was hypermethylated in osteosarcoma cells, and demethylase treatment increased the expression of miR-23a. The ectopic expression of miR-23a led to retarded proliferation, migration, and invasion of osteosarcoma cells, whereas the depletion of miR-23a resulted in the opposite effects. MiR-23a suppressed the transcription of RUNX2 and CXCL12 by binding to the 3' UTRs of these mRNAs. The cellular function of miR-23a is RUNX2/CXCL12-dependent, and the overexpression of RUNX2 or CXCL12 rescued the impaired cell growth, migration, and invasion induced by miR-23a. Nude mouse experiments indicated that miR-23a may inhibit the proliferation of osteosarcoma cells in vivo. Conclusion: We identified miR-23a as a tumor suppressor in osteosarcoma. Our data clarify the mechanism of osteosarcoma progression and demonstrated the potential for exploiting miR-23a as a diagnostic marker for osteosarcoma.

Previous studies have demonstrated that various microRNAs (miRNAs or miRs) are abnormally expressed in osteosarcoma (OS) and serve roles in its malignant development. An in-depth understanding of the specific roles of dysregulated miRNAs in OS may be important for cancer research and the identification of novel therapeutic targets. In the current study, reverse transcription-quantitative PCR was performed to determine miR-652 expression in OS tissues and cell lines. Cell Counting Kit-8 and Transwell invasion assays were used for assessing the effect of miR-652 on the proliferation and invasion of OS cells. Herein, miR-652 expression was assessed in OS and the effects and molecular mechanisms of miR-652 in OS cells were examined. The results revealed that miR-652 expression was significantly upregulated in OS tissues and cell lines compared with adjacent normal tissues and a normal human osteoblast cell line. Furthermore, miR-652 downregulation inhibited the proliferation and invasion of OS cells. miR-652 was also demonstrated to directly interact with the 3'-untranslated region of kruppel-like factor 9 (KLF9) and miR-652 negatively regulated KLF9 expression in OS cells. miR-652 and KLF9 mRNA levels were also revealed to be inversely correlated in OS tissues. Treatment with KLF9 small interfering RNA abolished the suppression of OS proliferation and invasion induced by miR-652 downregulation. miR-652 may serve an oncogenic role in OS cells by targeting KLF9 directly. The results also indicated that miR-652 may be an effective novel therapeutic target for the treatment of patients with OS.

The abnormal expression of microRNAs (miRNAs/miRs) has been observed in osteosarcoma (OS), and these differently expressed miRNAs contribute to the occurrence and development of OS by regulating various biological behaviours. Therefore, a comprehensive understanding of the detailed roles of aberrantly expressed miRNAs in OS progression may be favourable to the identification of promising therapeutic strategies for the treatment of patients with this malignancy. The present study demonstrated that miR‑660‑5p (miR‑660) expression was significantly upregulated in OS tissues and cell lines compared with that in normal adjacent tissues and normal human osteoblast hFOB1.19, respectively. miR‑660 downregulation led to a significant decrease in the proliferation and invasion of OS cells. Forkhead box O1 (FOXO1) was predicted as a potential target of miR‑660. The subsequent luciferase reporter assay indicated that miR‑660 directly binds to the 3'‑untranslated region of FOXO1. Furthermore, miR‑660 inhibition increased the FOXO1 expression in OS cells at mRNA and protein levels. Moreover, FOXO1 was downregulated in OS tissues and this downregulation was negatively correlated with miR‑660 levels. Besides, rescue experiments demonstrated that FOXO1 knockdown abolished the effects of miR‑660 knockdown on OS cell proliferation and invasion. These results suggest that miR‑660 may serve oncogenic roles in OS by directly targeting FOXO1. Targeting miR‑660 may be an effective candidate for the treatment of patients with OS.