Promotes Osteosarcoma Proliferation Research Articles

Exosomal Transfer of LCP1 Promotes Osteosarcoma Cell Tumorigenesis and Metastasis by Activating the JAK2/STAT3 Signaling Pathway

Increasing evidence indicates that lymphocyte cytosolic protein 1 (LCP1) overexpression contributes to tumor progression; however, its role in osteosarcoma (OS) remains unclear. We aimed to investigate the potential effect of LCP1 in OS and the underlying mechanisms. We first demonstrated that LCP1 is upregulated in OS cell lines and tissues. Then, we found that aberrant expression of LCP1 could induce the proliferation and metastasis of OS cells in vitro and in vivo by destabilizing neuregulin receptor degradation protein-1 (Nrdp1) and subsequently activating the JAK2/STAT3 signaling pathway. When coculturing OS cells with bone marrow-derived mesenchymal stem cells (BMSCs) in vitro, we validated that oncogenic LCP1 in OS was transferred from BMSCs via exosomes. Moreover, microRNA (miR)-135a-5p, a tumor suppressor, was found to interact upstream of LCP1 to counteract the pro-tumorigenesis effects of LCP1 in OS. In conclusion, BMSC-derived exosomal LCP1 promotes OS proliferation and metastasis via the JAK2/STAT3 pathway. Targeting the miR-135a-5p/LCP1 axis may have potential in treating OS.

Maternal embryonic leucine zipper kinase serves as a poor prognosis marker and therapeutic target in osteosarcoma.

Osteosarcoma is the most common primary malignancy of bones and frequently affects young children and adolescents. There are several challenges associated with treating osteosarcoma owing to the aggressiveness of the disease, as well as the risk of chemoresistance. Numerous studies are being performed with the aim of identifying improved prognostic and therapeutic markers for this malignancy. Maternal embryonic leucine zipper kinase (MELK) is an oncogene that has been studied in several types of cancer in recent years. In the present study, the expression of MELK in osteosarcoma and normal tissue samples was examined, and the effects of MELK expression on osteosarcoma cellular proliferation, metastasis, the cell cycle and apoptosis were demonstrated using CCK-8, wound healing, migration and invasion and apoptosis assays. The role of MELK in cancer progression in osteosarcoma was determined, revealing the association between MELK expression and prognosis of osteosarcoma. It was demonstrated that knockdown of MELK resulted in reduced proliferation, migration and invasion in vitro along with potentiation of apoptosis and cell cycle arrest. Furthermore, the effect of the targeted MELK inhibitor, OTSSP167, on tumor progression of osteosarcoma in vitro and in vivo was assessed. Mechanistically, it was demonstrated that MELK promoted osteosarcoma proliferation and metastasis by regulating PCNA and MMP9 expression via the PI3K/Akt/mTOR signaling pathway. Thus, the present study revealed the oncogenic role played by MELK, and established MELK as a valuable prognostic and therapeutic marker in osteosarcoma.

LncRNA SNHG4 promotes osteosarcoma proliferation and migration by sponging miR-377-3p.

BackgroundLong non‐coding RNAs (lncRNAs) have been identified as crucial regulatory factors in the occurrence and progression of osteosarcoma.MethodsQuantitative real‐time polymerase chain reaction was used for detecting small nucleolar RNA host gene 4 (SNHG4) and miR‐377‐3p in osteosarcoma cells and tissues. Kaplan–Meier method was applied for evaluating the association between SNHG4 expression and the overall survival of osteosarcoma patients. CCK8, EdU, flow cytometry, and transwell assay were performed to examine the cell proliferation, apoptosis, cycle, and migration of osteosarcoma cells.ResultsIn our study, we found that lncRNA SNHG4 was highly expressed in osteosarcoma tissues and cell lines. Additionally, the SNHG4 expression was related to distant metastasis, TNM stage, and survival of osteosarcoma patients. Through SNHG4 knockdown, the proliferation of osteosarcoma cells was considerably restrained and the cell apoptosis was induced in vivo and in vitro. Moreover, downregulated SNHG4 inhibited the cell migration and epithelial‐mesenchymal transition in HOS and MG63 cells. In mechanism, we found that SNHG4 acts as a competing endogenous RNA to sponge miR‐377‐3p, which is downregulated in osteosarcoma. Our results showed that there is a negative correlation between SNHG4 and miR‐377‐3p expression in osteosarcoma patients.ConclusionTaken together, SNHG4 promotes cell proliferation and migration by sponging miR‐377‐3p in osteosarcoma.

Overexpression of hsa_circ_0136666 predicts poor prognosis and initiates osteosarcoma tumorigenesis through miR-593-3p/ZEB2 pathway.

Background: Osteosarcoma (OS) is a type of malignant bone tumor with a growing incidence. Increasing studies indicate circular RNA (circRNA) has a vital function in tumorigenesis. Yet, how circRNA regulates OS development is not clear. In the present work, we aimed to investigate the roles of hsa_circ_0136666 in OS progression.Results: hsa_circ_0136666 was shown to be upregulated in OS and correlated with advanced stage and poor prognosis. Functional investigation using CCK8, colony formation assay and Transwell assay demonstrated that hsa_circ_0136666 promoted OS proliferation, migration and invasion, but inhibited cell death. Additionally, we identified hsa_circ_0136666 was a molecular sponge for miR-593-3p to facilitate ZEB2 expression. MiR-593-3p and ZEB2 were inversely expressed in OS tissues. And hsa_circ_0136666 exerts oncogenic roles in OS relying on miR-593-3p and ZEB2.Conclusion: Our results demonstrate the involvement of hsa_circ_0136666 in regulating OS tumorigenesis and it may be a therapeutic target.Methods: The expression of hsa_circ_0136666 was analyzed by qRT-PCR in OS tissues and cell lines. Proliferation was measured via CCK8 and colony formation assays. Migration and invasion were determined through Transwell assay. Luciferase reporter assay was utilized to determine the interaction between hsa_circ_0136666 and miR-593-3p or between miR-593-3p and ZEB2. Animal experiment was performed to investigate the role of hsa_circ_0136666 in vivo.

LncRNA CRNDE is activated by SP1 and promotes osteosarcoma proliferation, invasion, and epithelial-mesenchymal transition via Wnt/β-catenin signaling pathway.

Long noncoding RNAs (lncRNAs) were identified as a vital part in the development and progression of cancer in recent years. Colorectal neoplasia differentially expressed (CRNDE), a lncRNA, functions as an oncogene in some malignant neoplasias, but its role in the progression of osteosarcoma (OS) is still poorly understood. To dissect the difference in the expression of CRNDE, quantitative real-time polymerase chain reaction was utilized to evaluate it in OS tissues and cell lines (U2OS, MG63, and MNNG/HOS) compared with that in the adjacent normal tissues/osteoblast cells (hFOB1.19). The role of CRNDE in OS lines was assessed using Cell Counting Kit-8, colony formation, 5-ethynyl-2'-deoxyuridine staining, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling staining, flow cytometry, Transwell assays, and Western blot, respectively. The results demonstrated that the expression of CRNDE was high in OS tissues and cell lines, and partly induced by SP1. CRNDE knockdown attenuated OS cell proliferation and invasion and induced apoptosis and G0/G1 arrest. Moreover, the expression of mesenchymal markers N-cadherin, Vimentin and Snail were downregulated, while the expression of epithelial markers E-cadherin and ZO-1 were conversely upregulated due to CRNDE knockdown. The mechanistic investigations showed that CRNDE promoted glycogen synthase kinase-3β phosphorylation to activate the Wnt/β-catenin pathway. The results suggested that lncRNA CRNDE indeed contributed to OS proliferation, invasion, and epithelial-mesenchymal transition, working as an oncogene, demonstrating that lncRNA CRNDE may be a valid therapeutic target for the OS.

The Notch Pathway Promotes Osteosarcoma Progression through Activation of Ephrin Reverse Signaling.

Despite significant advancements in the diagnosis and treatment of osteosarcoma, the molecular mechanisms underpinning disease progression remain unclear. This work presents strong clinical and experimental evidence demonstrating that Notch signaling contributes to osteosarcoma progression. First, using a cohort of 12 patients, Notch genes were upregulated in tumors compared with adjacent normal tissue, and high tumor expression of Notch1 intercellular domain (NICD1) and the Notch target gene Hes1 correlated with poor chemotherapy response. Data mining of publicly available datasets confirmed that expression of Notch pathway genes is related to poor prognosis in osteosarcoma. On the basis of in vitro analysis, Notch signaling promoted osteosarcoma proliferation, enhanced chemoresistance, facilitated both migration and invasion, and upregulated stem cell-like characteristics. Xenograft models demonstrated that Notch signaling promotes primary tumor growth and pulmonary metastasis, and Notch inhibition is effective in reducing tumor size and preventing metastasis. Mechanistically, activated Notch signaling induces the expression of ephrinB1 and enhances the tumor-promoting ephrin reverse signaling. Overall, these findings provide functional evidence for Notch pathway genes as candidate biomarkers to predict prognosis in patients with osteosarcoma, and suggest a mechanistic rationale for the use of Notch inhibitors to treat osteosarcoma. IMPLICATIONS: The study provides preclinical evidence for Notch pathway as a molecular marker to predict osteosarcoma prognosis and as a therapeutic target against osteosarcoma. In addition, we identified a novel mechanism that ephrin reverse signaling acts as a key mediator of Notch pathway.

Long non-coding RNA MNX1-AS1 promoted osteosarcoma proliferation and invasion via inhibiting KISS1.

Recent studies revealed that long non-coding RNAs (lncRNAs) participate in the progression and development of many human diseases. In this work, we are committed to uncovering the association between lncRNA MNX1-AS1 and the development of osteosarcoma. MNX1-AS1 expression of osteosarcoma cells and tissue samples was detected by Real Time-quantitative Polymerase Chain Reaction (RT-qPCR). Besides, we conducted functional assays including cell proliferation assay, colony formation, and transwell assays. Furthermore, the underlying mechanism including RT-qPCR and Western blot assay was performed. MNX1-AS1 was higher-expressed in osteosarcoma samples than that in adjacent tissues. The abilities of proliferation and invasion were suppressed after MNX1-AS1 was knocked down in vitro. Moreover, KISS1 expression was upregulated at mRNA and protein level via silence of MNX1-AS1. Furthermore, the underlying mechanism of the development of osteosarcoma were investigated by RT-qPCR and Western blot assay. Our study demonstrated that MNX1-AS1 could enhance osteosarcoma cell proliferation and invasion by inhibiting KISS1, which might contribute to the therapy for osteosarcoma.

CircSAMD4A accelerates cell proliferation of osteosarcoma by sponging miR-1244 and regulating MDM2 mRNA expression

Here, we firstly examined the proliferation and invasion capacities of different osteosarcoma (OS) cell lines, and analyzed the profiling of the differentially expressed circular RNAs(circRNAs). Then, we identified a novel circRNA(circ_101356/circ_0004846, we named circSAMD4 in the present study) and found it was highly expressed in the OS tissues compared to adjacent non-cancerous tissues. Furthermore, we indicated that circSAMD4A promoted OS proliferation in vivo and in vitro. In addition, we proved that circSAMD4A enhances osteosarcoma cells stemness features. In mechanism, circSAMD4A could sponge miR-1244 in OS cells. Moreover, we verified that MDM2 was a target of miR-1244. In conclusion, circSAMD4A/miR-1244/MDM2 regulatory loop might be a promising therapeutic target for OS treatment.

Long noncoding RNA AFAP1-AS1 promoted osteosarcoma proliferation and invasion via upregulating BDNF.

Recently, long noncoding RNAs (lncRNAs) have got much attention for their role in tumor progression. LncRNA AFAP1-AS1 was studied in this research to identify how it affects the development and proliferation of osteosarcoma. The mRNA expression of AFAP1-AS1 in osteosarcoma cells and tissue samples was detected by quantitative real-time polymerase chain reaction (qRT-PCR). Moreover, cell proliferation assay and Matrigel assay were performed. Furthermore, the underlying mechanism was explored by using qRT-PCR and Western blot assay. The expression level of AFAP1-AS1 was higher in osteosarcoma samples than that in adjacent tissues. In addition, the proliferation and invasion were inhibited after AFAP1-AS1 was downregulated in vitro. Besides, the mRNA and protein expression levels of brain derived neurotrophic factor (BDNF) were reduced after downregulation of AFAP1-AS1. Furthermore, the expression level of BDNF was positively related to the expression of AFAP1-AS1 in osteosarcoma tissues. AFAP1-AS1 could enhance the proliferation and invasion of osteosarcoma cells by upregulating BDNF, which might be a potential therapeutic target in osteosarcoma.

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

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

Macrophage-derived CCL18 promotes osteosarcoma proliferation and migration by upregulating the expression of UCA1.

Osteosarcoma (OS), which is the most common primary malignant bone tumor, has a high incidence of pulmonary metastasis. CCL18 (C-C motif chemokine ligand 18), which is secreted by tumor-associated macrophages (TAMs), has been found to be increased in various tumors and is associated with tumor metastasis. However, the role of CCL18 in OS remains unclear. Here, we evaluated the effect of CCL18 on the OS cell lines MG63 and 143B and explored its potential mechanisms. We found that CCL18 enhanced the proliferation and migration of OS cells and upregulated UCA1 through transcription factor EP300. Subsequently, we further revealed that the downstream Wnt/β-catenin signaling pathway participated in this process. In addition, the high expression of CCL18 in both tissue and serum from patients was closely related to pulmonary metastasis and poor survival in OS patients. The tumor xenograft models also showed that CCL18 promoted the metastasis of OS cells. Collectively, our study indicated that macrophage-derived CCL18 promotes OS proliferation and metastasis via the EP300/UCA1/Wnt/β-catenin pathway and that CCL18 may be used as a prognostic marker and therapeutic target of OS. KEY MESSAGES: CCL18 promotes proliferation and migration of osteosarcoma cells by EP300/ UCA1/ Wnt/β-catenin pathway. CCL18+ TAMs are significantly correlated with pulmonary metastasis and poor survival in osteosarcoma patients. CCL18 may be used as a prognostic marker and therapeutic target for osteosarcoma.

Nucleoside diphosphate kinase B promotes osteosarcoma proliferation through c-Myc

ABSTRACTOsteosarcoma (OS) is one of the most common primary bone tumors and has a high disablity rate and case-fatality rate. The protracted stagnancy of the chemotherapy program and surgical technology for OS treatment prompted us to focus on the mechanisms of cancer carcinogenesis progression in OS.Nucleoside diphosphate kinase B (NME2) is a type of nucleoside diphosphate kinase that plays an important role in cellular processes. In this study, we report overexpression of NME2 in OS cell lines and correlate this overexpression with the clinicopathologic features of osteosarcoma. We used si-NME2 to downregulate expression of NME2 in OS cell lines. The results of the CCK8 and clone forming assays show that NME2 promotes OS cell line proliferation. Western blot assays show that deregulation of NME2 results in enhanced the expression of c-Myc, which promotes OS proliferation.

Long non-coding RNA CCAT1/miR-148a axis promotes osteosarcoma proliferation and migration through regulating PIK3IP1.

Osteosarcoma is the most frequent type of malignant primary bone tumor. Although many efforts have been made, the survival rate of osteosarcoma still remains unsatisfied. Long non-coding RNAs (lncRNAs) have been demonstrated to be associated with many diseases including tumors, and involved in the regulation of a wide array of pathophysiological processes. Colon-cancer-associated transcript-1 (CCAT1) was first identified in colon cancer and has subsequently been reported to perform many functions in tumor progression. The present study aimed to comprehensively explore the biological functions of CCAT1 and its underlying mechanism in osteosarcoma cells. Our findings revealed that CCAT1 was upregulated in osteosarcoma tissues and cells, and was involved in the proliferation and migration of osteosarcoma via regulating miR-148a/phosphatidyl inositol 3-kinase interacting protein 1 (PIK3IP1) signal pathway.

A novel mechanism of mTORC1-mediated serine/glycine metabolism in osteosarcoma development

Osteosarcoma is the major malignant primary bone cancer in children and adolescents, which is highly aggressive with frequent acquisition of chemoresistance phenotypes. Although much progress has been made, mechanisms of osteosarcoma rapid growth and chemoresistance are still not well elucidated. Generally, alternated metabolic characterization has been proposed to be a hallmark of cancer, yet it is lack of a systematic characterization of cancer metabolic networks. In the present study, we aim to characterize osteosarcoma metabolism and key regulators to reveal mechanisms of how osteosarcoma grows and resists apoptosis under stress conditions. The results demonstrate that mTORC1 pathway is hyperactivated in clinical osteosarcoma samples. However, inhibition of mTORC1 may not be enough to induce significant death of osteosarcoma cells. Results of GC-TOFMS suggested that inhibition of mTORC1 reduce one-carbon amino acids, serine and glycine, in osteosarcoma cells. Moreover, mTORC1 regulates serine/glycine de novo synthesis via modulating glycolysis and serine/glycine synthesis gene expressions. Further, mTORC1/serine/glycine metabolic axis promotes osteosarcoma proliferation and antioxidant ability to environmental stress, which finally leads to cell survival. Our results identify a novel mechanism of mTORC1-mediated serine/glycine metabolism as a significant protective system in osteosarcoma cells.

Silencing of Eag1 Gene Inhibits Osteosarcoma Proliferation and Migration by Targeting STAT3-VEGF Pathway.

So far, the role of Ether à go-go 1 (Eag1) potassium channels in migration and invasion progression of cancers remains elusive. In the present study, the effects of Eag1 knockdown on osteosarcoma cell proliferation, growth, and apoptosis were examined. Then, we evaluated the effects of Eag1 silencing on osteosarcoma cell migration and invasion. In addition, we detected the expression of vascular endothelial growth factor (VEGF) and signal transducer and activator of transcription 3 (STAT3) in osteosarcoma cell treated with Eag1 small interfering RNAs (siRNAs). Finally, STAT3 siRNA was employed to determine the influence of downregulation of STAT3 on cell proliferation and migration. The results showed that knockdown of Eag1 significantly suppressed osteosarcoma cell proliferation and osteosarcoma xenografts growth. However, Eag1 silencing had little effect on cell apoptosis. Additionally, osteosarcoma cell adhesion, migration, and invasion were also potently attenuated. Notably, the expression levels of VEGF decreased evidently upon Eag1 siRNAs treatment, paralleled with reductions in the expression levels of STAT3. Moreover, a similar pattern was observed in osteosarcoma cell proliferation and migration suppression between STAT3 siRNA and Eag1 siRNAs groups. Our data indicated that Eag1 promotes osteosarcoma proliferation and migration, at least in part, by targeting STAT3-VEGF pathway.