HOTAIR promotes osteosarcoma development by sponging miR-217 and targeting ZEB1

Long noncoding RNAs (lncRNAs) have drawn increasing attention because of the role which they play in various diseases, including osteosarcoma. So far, the function and mechanism of HOTAIR in osteosarcoma are unclear. In our study, we observed that HOTAIR was elevated accompanied with a decrease of miR-217 and an increase of ZEB1 in human osteosarcoma cells including U2OS, MG63, Saos-2, and SW1353 compared with human osteoblast cell line hFOB. In addition, the subsequent functional assay exhibited that silencing HOTAIR could significantly repress osteosarcoma cell growth, migration, invasion, and induce cell apoptosis capacity, which indicated that HOTAIR exerted an oncogenic role in osteosarcoma. Moreover, it was revealed by using bioinformatics analysis that HOTAIR can be targeted by microRNA-217 (miR-217). miR-217 has been recognized as a crucial tumor suppressive gene in cancers. We verified that mimics of miR-217 were able to suppress the osteosarcoma development. Furthermore, real-time quantitative PCR showed that HOTAIR siRNA increased miR-217 expression. Besides these, ZEB1 was identified as a downstream gene of miR-217 and we found that HOTAIR can mediate osteosarcoma progress by upregulating ZEB1 expression via acting as a competitive endogenous RNA (ceRNA) via miR-217. Taken these together, our findings in this study indicated that HOTAIR/miR-217/ZEB1 axis, as a novel research point can provide new insights into molecular mechanism of osteosarcoma development.     

Dysregulated circRNA_100876 suppresses proliferation of osteosarcoma cancer cells by targeting microRNA-136

The aim of this study was to explore the regulatory mechanism of circRNA_100876/ microRNA-136 (miR-136) axis in the development and progression of osteosarcoma cancer. Quantitative real-time polymerase chain reaction (RT-qPCR) was used to evaluate the expression levels of circRNA_100876 and miR-136 in osteosarcoma cancer samples and the adjacent nontumor tissues. Then, cell proliferation, cell cycle, apoptosis, and migration of circRNA_100876-knocked down cells were analyzed by in vitro and in vivo experiments, using cell counting kit-8 (CCK-8), flow cytometry, and transwell and tumorigenesis assays. The expression of circRNA_100876 was found to be significantly upregulated in osteosarcoma, and was closely correlated with the tumor size and tumor differentiation degree. In addition, the knockdown of circRNA_100876 could significantly inhibit the tumor growth, both in vitro and in vivo. Flow cytometry and Western blot analysis results showed that the downregulation of circRNA_100876 inhibited osteosarcoma cells proliferation via promoting apoptosis and arresting more cells in the G2/M stage, as suggested by the expression of apoptosis and cell cycle pathway-related proteins, which changed consistently. Furthermore, the level of miR-136 was negatively correlated with the expression of circRNA_100876, and miR-136 inhibitors were able to reverse the suppression of cell proliferation induced by silencing circRNA_100876. Our study demonstrates that the dysregulation of circRNA_100876 could induce apoptosis and arrest the cell cycle at G2/M stage, followed by suppression of cell proliferation in osteosarcoma, while silencing miR-136 could restore the cell growth. Therefore, circRNA_100876 might serve as a promising biomarker and treatment target for osteosarcoma.     

Feedback modulation of endothelial cells promotes epithelial-mesenchymal transition and metastasis of osteosarcoma cells by Von Willebrand Factor release

Endothelial cells (ECs), as a tumor niche cell, generate and secrete Von Willebrand factor (VWF) that is linked to osteosarcoma (OS) progression. However, the role and regulatory mechanisms of VWF that underpin OS progression remain unclear. Here, using a coculture system ex vivo, we showed that ECs promoted the epithelial-mesenchymal transition (EMT) process in OS cells via enhanced VWF secretion. VWF secreted by ECs directly contributed to OS EMT and metastasis by activating NF-κB signaling. In addition, OS cells exerted a feedback effect on ECs to promote VWF release via activation of phospholipase D 1 signaling, through which enhanced VWF secretion results in further tumor deterioration. To conclude, ECs served as a modulator and an effector of OS, accelerating OS exacerbation by VWF release.     

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.     

SIRT1-ZEB1-positive feedback promotes epithelial-mesenchymal transition process and metastasis of osteosarcoma

Osteosarcoma is the most common malignant bone cancer that mainly affects children and young adults. Recently, the NAD⁺-dependent deacetylase, sirtuin 1 (SIRT1), has been reported to play a key role in the development of malignant tumors. The study aimed to investigate the role of SIRT1 in osteosarcoma and explore its underlying oncogenic mechanisms. The prognostic value of SIRT1 in osteosarcoma was assessed through detection of SIRT1 expression based on osteosarcoma biopsy tissue. Then, to further investigate the effect of SIRT1 in osteosarcoma, osteosarcoma cells were treated with small interfering RNA SIRT1 and overexpressed SIRT1 to detect the cell migration, invasion, and epithelial-mesenchymal transition (EMT). The levels of SIRT1 expression were significantly higher in osteosarcoma tissues than those in adjacent normal tissues, and the SIRT1 protein level may be coupled with metastatic and poor prognosis risk in patients with osteosarcoma. Moreover, SIRT1 silencing inhibited the migration as well as invasion ability of osteosarcoma cells in vitro, and SIRT1 upregulation reversed those effects. Finally, we found that SIRT1-ZEB1-positive feedback enhanced the EMT process and metastasis of osteosarcoma. Altogether, the results of the current study revealed that high levels of SIRT1 might be a biomarker for a high metastatic rate in patients with osteosarcoma, which suggested that inhibition of SIRT1 might be promising for the therapeutics of osteosarcoma.     

Apelin promotes osteosarcoma metastasis by upregulating PLOD2 expression via the Hippo signaling pathway and hsa_circ_0000004/miR-1303 axis

Osteosarcoma is a highly mortal bone tumor, with a high metastatic potential, promoted in part by the enzyme procollagen-lysine 2-oxoglutarate 5-dioxygenase 2 (PLOD2). Increasing level of PLOD2 in osteosarcoma tissue correlates with lymphatic and distant metastasis. The adipokine apelin (APLN) is also found in different cancers and APLN upregulation promotes angiogenesis and metastasis, but its effects on osteosarcoma metastasis are uncertain. We explored APLN functioning in metastatic osteosarcoma. An analysis of records from the Gene Expression Omnibus (GEO) database showed higher levels of APLN expression in osteosarcoma tissue than in normal tissue. Similarly, levels of APLN and PLOD2 mRNA synthesis were upregulated in osteosarcoma tissue. Levels of APLN and PLOD2 protein correlated positively with osteosarcoma clinical stages. APLN increased PLOD2 expression in human osteosarcoma cell lines and cell migration via the mammalian Sterile 20-like kinase 1 (MST1), monopolar spindle-one-binder protein (MOB)1, and YAP cascades, and through hsa_circ_0000004 functioning as a sponge of miR-1303. We also found that knockdown of APLN antagonized lung metastasis in mice with osteosarcoma. APLN may be a therapeutic target in osteosarcoma metastasis.     

MicroRNA-1296-5p suppresses the proliferation,migration, and invasion of human osteosarcoma cells by targeting NOTCH2

Osteosarcoma (OS) is a highly aggressive bone tumor with a poor prognosis. MicroRNAs are revealed to exerts essential roles in the carcinogenesis and tumor invasion of OS. But, the function of miR-1296-5p and its related mechanism in OS progression have not yet been studied. This study discovered the levels of miR-1296-5p in OS and corresponding noncancerous tissues, and we demonstrated that miR-1296-5p level was markedly downregulated in tumor specimens as compared with nontumor tissues. In addition, we discovered that miR-1296-5p was also underexpressed in OS cells compared with the hFOB1.19 osteoblast cells. Interestingly, the reduced expression of miR-1296-5p was confirmed to associated with large tumor size, advanced tumor stages, and distance metastasis, respectively. Patients with OS low-expressing miR-1296-5p showed a prominent shorter survival. In addition, gain-of-function assays verified that miR-1296-5p overexpression remarkably repressed OS cell proliferation, migration, and invasion. Conversely, depletion of miR-1296-5p facilitated the growth and mobility of OS cells. Notably, miR-1296-5p inversely modulated notch receptor 2 (NOTCH2) in OS cells. The level of NOTCH2 messenger RNA was negatively correlated with miR-1296-5p level in OS samples. NOTCH2 knockdown markedly suppressed the abilities of MG-63 cell proliferation and mobility. More importantly, the restoration of NOTCH2 prominently rescued miR-1296-5p-induced tumor-suppressive effects on MG-63 cells. In conclusion, our study identified the reduced expression of miR-1296-5p, which contributed to OS progression. miR-1296-5p might be a promising prognostic marker and therapeutic target in OS.     

Circular RNA hsa_circ_0000658 inhibits osteosarcoma cell proliferation and migration via the miR-1227/IRF2 axis

Osteosarcoma (OS) is the most frequently occurring bone cancer. Circular RNAs (circRNAs) have been shown to exert pivotal impact in modulation of gene expression, but their roles in OS are still not fully understood. In this study, we analysed the role of circ-0000658 in OS. Thereafter, molecular techniques such as Western blot, qRT-PCR, RNA-binding protein immunoprecipitation and Dual-Luciferase reporter assays were implemented to investigate the role of circ-0000658/miR-1227/interferon regulatory factor-2 (IRF2) axis in OS. Eventually, the impact of circ-0000658 on tumour growth and metastasis was observed in a xenograft mouse model. The results of this study revealed that circ-0000658 exhibits low levels in OS tissues and cell lines. Moreover, circ-0000658 repression promoted cell cycle, proliferation, invasion and migration but inhibited the apoptosis of OS cells. Researches have previously shown that circ-0000658 contains a binding site for miR-1227 and thus can abundantly sponge miR-1227 to up-regulate the expression of its target gene IRF2. Moreover, both inhibition of miR-1227 and overexpression of IRF2 reversed cell proliferation and invasion, which was triggered by circ-0000658 repression. Conclusively, circ-0000658 modulates biological function of OS cells through the miR-1227/IRF2 axis. Therefore, circ-0000658 might act as a possible novel therapeutic target for the treatment of OS.     

miR181c promotes apoptosis and suppresses proliferation of metanephric mesenchyme cells by targeting Six2 in vitro

Increasingly recognized importance has been assumed for microRNA (miRNA) in the regulation of the delicate balance of gene expression. In our study, we aimed to explore the regulation role of miR181c towards Six2 in metanephric mesenchyme (MM) cells. Bioinformatics analysis, luciferase assay and semi‐quantitative real‐time (RT) PCR, subsequently RT PCR, Western blotting, 5‐ethynyl‐2′‐deoxyuridine cell proliferation assay, Cell Counting Kit‐8 assay, immunofluorescence and flow cytometry, were employed to verify the modulation function of miR181c on Six2 in the mK3 MM cell line that is one kind of MM cells. miR181c was predicted to bind the 3′ untranslated region of Six2 by bioinformatics analysis, which was subsequently validated by the in vitro luciferase reporter assay. Moreover, transfection of miR181c mimic can decrease the expression of Six2 both in mRNA and protein levels in mK3 cells. Still, ectopic expression of miR181c inhibits the proliferation, promotes the apoptosis and even makes the nephron progenitor phenotype lose mK3 cells. These results revealed the ability of a single miRNA–miR181c to downregulate the expression of Six2, restrain the proliferation and promote the apoptosis that even makes the nephron progenitor phenotype lose MM cells, suggesting a potential role of miR181c during the kidney development. Copyright © 2014 John Wiley & Sons, Ltd.     

LncRNA NBR2 inhibits epithelial-mesenchymal transition by regulating Notch1 signaling in osteosarcoma cells

Long noncoding RNAs (lncRNAs) have been identified to have increasingly important roles in tumorigenesis, and they may serve as novel biomarkers for cancer therapy. Recent studies have demonstrated that lncRNA NBR2 (neighbor of BRCA1 gene 2), a novel identified lncRNA, is decreased in several cancers; however, the role of NBR2 in the development of osteosarcoma has not been elucidated. In our study, we found that NBR2 expression was downregulated in osteosarcoma tissues, and osteosarcoma cases with lower NBR2 expression exhibited a shorter overall survival time compared with those with higher NBR2 expression. NBR2 overexpression inhibited osteosarcoma cell proliferation, invasion, and migration but did not increase apoptosis. Furthermore, RNA-binding protein immunoprecipitation assays confirmed that NBR2 directly binds to Notch1 protein. Furthermore, overexpression of Notch1 in NBR2-overexpressing osteosarcoma cells reversed the effects of NBR2 on cell proliferation, invasion, migration, and epithelial-mesenchymal transition. The in vivo results showed that NBR2 overexpression inhibited tumor growth in nude mice that were inoculated with osteosarcoma cells. NBR2 overexpression also suppressed the messenger RNA (mRNA) expression of Notch1, N-cadherin, and vimentin and increased the mRNA expression of E-cadherin in the tumor tissues. These data indicated that NBR2 served as a tumor suppressor gene in osteosarcoma and inhibited osteosarcoma cell proliferation, invasion, and migration. The current study provides a novel insight and treatment strategy for osteosarcoma.     

miR-15a-5p suppresses inflammation and fibrosis of peritoneal mesothelial cells induced by peritoneal dialysis via targeting VEGFA

Long-term peritoneal dialysis (PD) often ends up with ultrafiltration failure (UFF) which is partially caused by persistent inflammation and fibrosis of peritoneal tissues. However, the mechanism is still unclear. In the current study, the peritoneum from UFF patients demonstrated inflammation and fibrosis which were positively related to the expression of vascular endothelial growth factor A (VEGFA). The in vitro model using human peritoneal mesothelial cells (HPMCs) stimulated by high glucose or advanced glycation end (AGE) product showed consistent changes of inflammation, fibrosis, and VEGFA. What's more, we showed that VEGFA was an instigator of inflammation and fibrosis. Several microRNAs (miRNAs) have been reported to regulate expression of VEGFA elsewhere. Five of them were selected to test the expression in the peritoneum of patients with PD. Results suggested that miR-15a-5p was the most significantly downregulated one. Also, in high glucose or AGE product-stimulated HPMCs, miR-15a-5p decreased. When miRNA mimic was used to restore the expression of miR-15a-5p, high glucose-induced VEGFA was repressed. The predicted binding site between these two molecules was confirmed by the dual-luciferase assay. Restoration of miR-15a-5p restrained inflammation and fibrosis of HPMCs. TGF-β1/Smad2 was shown to be the downstream signaling pathway and their activity was regulated by miR-15a-5p/VEGFA. In conclusion, our current study demonstrates that miR-15a-5p acts as a regulator of VEGFA mRNA and the following inflammation and fibrosis in peritoneal mesothelial cells. The miR-15a-5p/VEGFA pathway may be a potential target for preventing ultrafiltration failure in patients with PD.     

miR-203 accelerates apoptosis and inflammation induced by LPS via targeting NFIL3 in cardiomyocytes

Myocarditis is an inflammatory disease of the myocardium. MicroRNA-203 (miR-203) is involved in various physiological and pathological processes. In this work, we aimed to explore the roles and potential mechanisms of miR-203 in myocarditis in vitro. Cardiomyocyte H9c2 was subjected to 10 μg/mL lipopolysaccharide (LPS) for 24 hours. Real-time polymerase chain reaction analysis revealed that LPS upregulated miR-203 expression in H9c2 cells. Cell counting kit-8 (CCK-8) and lactate dehydrogenase (LDH) assays demonstrated that inhibition of miR-203 reduced cell injury induced by LPS. The cell apoptosis rate, caspase 3 activity, caspase 3/7 activities, and the expression of cleaved-caspase 3 (c-caspase 3) were declined upon miR-203 depletion. In addition, miR-203 silencing attenuated the expression and production of inflammatory cytokines (tumor necrosis factor-α, interleukin [IL]-6, and IL-8). On the contrary, overexpression of miR-203 showed the opposite trend in cell apoptosis and inflammation. Luciferase reporter assay confirmed that miR-203 could bind with the nuclear factor interleukin-3 (NFIL3) 3′-untranslated regions (3′-UTR), and miR-203 regulated the expression of NFIL3 negatively. Moreover, NFIL3 silencing partly abolished the myocardial protective functions of miR-203 inhibitor. Herein, we suggest that miR-203 promoted cell apoptosis and inflammation induced by LPS via targeting NFIL3.