Qingcheng Yang

DEC2 expression is positively correlated with HIF-1 activation and the invasiveness of human osteosarcomas

BackgroundOsteosarcoma is the most common malignancy of bone. HIF-1 (hypoxia-inducible factor 1) activation is critical for the metabolic reprogramming and progression of solid tumors, and DEC2 (differentiated embryonic chondrocyte gene 2) has been recently reported to suppress HIF-1 in human breast and endometrial cancers. However, the roles of HIF-1 and DEC2 in human osteosarcomas remain unclear.MethodsWe evaluated the correlation of DEC2 and HIF-1 expression to the prognosis, and studied the roles of DEC2 and HIF-1 activation in the invasiveness of osteosarcoma. Multiple approaches including immunohistochemical staining of clinical osteosarcoma tissues, siRNA-based knockdown and other molecular biology techniques were used. Particularly, by using a repetitive trans-well culture-based in vitro evolution system, we selected a more invasive subpopulation (U2OS-M) of osteosarcoma cells from U2OS and used it as a model to study the roles of DEC2 and HIF-1 in the invasiveness of osteosarcoma.ResultsWe found that the expression of DEC2 was positively correlated with HIF-1α levels, and HIF-1α expression positively correlated with poor prognosis in osteosarcomas. DEC2 knockdown in osteosarcoma cell lines (U2OS, MNNG and 143B) attenuated HIF-1α accumulation and impaired the up-regulation of HIF-1 target genes in response to hypoxia. Compared with the low invasive parental U2OS, U2OS-M showed higher levels of DEC2 expression which were confirmed at both mRNA and protein levels. Importantly, we found that the increased DEC2 expression resulted in a more rapid accumulation of HIF-1α in U2OS-M cells in response to hypoxia. Finally, we found that HIF-1 activation is sufficient to upregulate DEC2 expression in osteosarcoma cells.ConclusionTaken together, whereas DEC2 was found to promote HIF-1α degradation in other types of tumors, our data indicate that DEC2 facilitates HIF-1α stabilization and promotes HIF-1 activation in osteosarcoma. This implies that DEC2 may contribute to the progression and metastasis of human osteosarcoma by sensitizing tumor cells to hypoxia. On the other hand, HIF-1 activation may contribute to the expression of DEC2 in osteosarcoma. This is the first demonstration of a novel DEC2-HIF-1 vicious cycle in osteosarcoma and a tumor-type specific role for DEC2.

GSK3β negatively regulates HIF1α mRNA stability via nucleolin in the MG63 osteosarcoma cell line

Hypoxia-inducible factor 1α (HIF1α) is a transcription factor involved in the growth, invasion and metastasis of malignant tumors. Glycogen synthase kinase 3 beta (GSK3β) is a protein kinase involved in a variety of signaling pathways, such as the Wnt and NF-κB pathways; this kinase can affect tumor progress through the regulation of transcription factor expression and apoptosis. Recent studies showed that GSK3β was involved in the expression of HIF1α. However, the effect of GSK3β on HIF1α expression in osteosarcoma cells remains unknown. To understand the relationship between GSK3β and HIF1α comprehensively, small RNA interference techniques, Western blot analyses, quantitative real-time PCR analyses and luciferase assays were used in our study. Experimental data revealed that inhibition of GSK3β could increase HIF1α protein levels and expression of its target genes by increasing the stability of the HIF1α mRNA, not by affecting the HIF1α protein stability, and that this process could be mediated by nucleolin.

Overexpression of X-Box Binding Protein 1 (XBP1) Correlates to Poor Prognosis and Up-Regulation of PI3K/mTOR in Human Osteosarcoma

Increasing evidence demonstrates that dysregulation of XBP1 function contributes to tumorigenesis in some cancers. However, little is known about the role of XBP1 in the progression of osteosarcoma (OS). The expression of XBP1 in OS samples was measured by quantitative RT-PCR and Western blotting assays. Cell cycle analysis and cell counting kit 8 (CCK8) assays were performed to determine the effects of XBP1 expression on cells growth capacity. Cell apoptosis coassay was applied to determine cell survival. The expression of genes affected by XBP1 was examined by quantitative RT-RCR and validated by Western blotting assays. XBP1 was overexpressed in OS clinical samples compared with corresponding non-cancerous tissues. Overexpression of XBP1 was significantly associated with advanced clinical stages, high degree of malignancy and low tumor necrosis rate. Furthermore, hypoxia activated XBP1, and silencing XBP1 significantly enhanced OS cell apoptosis. Knock-down of XBP1 resulted in inhibition of OS growth. Most importantly, knockdown of XBP1 led to down-regulation of PIK3R3 and mTOR. Taken together, XBP1 is up-regulated and has a pro-tumor effect in OS with activation of PI3K/mTOR signaling. Thus, targeting XBP1 may provide a new potential therapeutic method for OS.

MiR-542-5p is a negative prognostic factor and promotes osteosarcoma tumorigenesis by targeting HUWE1

Recent evidence has demonstrated that microRNAs (miRNAs) are involved in the proliferation and metastasis of osteosarcoma. Using miRNA microarray and functional screening methods to compare miRNA expression profiles in osteosarcoma cell lines treated with Trichostatin A (TSA), overexpression of miR-542-5p was determined to be involved in the proliferation of osteosarcoma. We used isobaric tags for relative and absolute quantitation (iTRAQ) and nanoscale liquid chromatography-mass spectrometry (NanoLC−MS/MS) to identify differentially expressed proteins in MNNG/HOS and U2OS osteosarcoma cell lines transfected with miR-542-5p; in both cell lines, seven proteins were downregulated, and nine were upregulated. HUWE1 was found to be a direct target of miR-542-5p in both osteosarcoma cell lines, and was negatively correlated with miR-542-5p levels in human osteosarcoma tissues. Moreover, the expression of miR-542-5p was upregulated in human osteosarcoma tissue compared with non-tumor adjacent tissue. Kaplan-Meier analysis revealed that overexpression of miR-542-5p predicted poor prognosis for osteosarcoma patients. Taken together, our results indicated that miR-542-5p plays a critical role in the proliferation of osteosarcoma and targets HUWE1.

High Expression of XRCC6 Promotes Human Osteosarcoma Cell Proliferation through the β-Catenin/Wnt Signaling Pathway and Is Associated with Poor Prognosis

Increasing evidences show that XRCC6 (X-ray repair complementing defective repair in Chinese hamster cells 6) was upregulated and involved in tumor growth in several tumor types. However, the correlation of XRCC6 and human osteosarcoma (OS) is still unknown. This study was conducted with the aim to reveal the expression and biological function of XRCC6 in OS and elucidate the potential mechanism. The mRNA expression level of XRCC6 was measured in osteosarcoma cells and OS samples by quantitative transcription-PCR (qRT-PCR). The expression of XRCC6 protein was measured using Western blot and immunohistochemical staining in osteosarcoma cell lines and patient samples. Cell Counting Kit 8 (CCK8), colony-forming and cell cycle assays were used to test cell survival capacity. We found that XRCC6 was overexpressed in OS cells and OS samples compared with the adjacent non-tumorous samples. High expression of XRCC6 was correlated with clinical stage and tumor size in OS. Reduced expression of XRCC6 inhibits OS cell proliferation through G2/M phase arrest. Most importantly, further experiments demonstrated that XRCC6 might regulate OS growth through the β-catenin/Wnt signaling pathway. In conclusion, these findings indicate that XRCC6 exerts tumor-promoting effects for OS through β-catenin/Wnt signaling pathway. XRCC6 may serve as a novel therapeutic target for OS patients.

CSE1L interaction with MSH6 promotes osteosarcoma progression and predicts poor patient survival

To discover tumor-associated proteins in osteosarcoma, a quantitative proteomic analysis was performed to identify proteins that were differentially expressed between osteosarcoma and human osteoblastic cells. Through clinical screening and a functional evaluation, chromosome segregation 1-like (CSE1L) protein was found to be related to the growth of osteosarcoma cells. To date, little is known about the function and underlying mechanism of CSE1L in osteosarcoma. In the present study, we show that knockdown of CSE1L inhibits osteosarcoma growth in vitro and in vivo. By co-immunoprecipitation and RNA-seq analysis, CSE1L was found to interact with mutS homolog 6 (MSH6) and function as a positive regulator of MSH6 protein in osteosarcoma cells. A rescue study showed that decreased growth of osteosarcoma cells by CSE1L knockdown was reversed by MSH6 overexpression, indicating that the activity of CSE1L was an MSH6-dependent function. In addition, depletion of MSH6 hindered cellular proliferation in vitro and in vivo. Notably, CSE1L expression was correlated with MSH6 expression in tumor samples and was associated with poor prognosis in patients with osteosarcoma. Taken together, our results demonstrate that the CSE1L-MSH6 axis has an important role in osteosarcoma progression.

SLC3A2 is upregulated in human osteosarcoma and promotes tumor growth through the PI3K/Akt signaling pathway

Growing evidence indicates that SLC3A2 (solute carrier family 3 member 2) is upregulated and correlates with tumor growth in multiple types of cancers, while the role of SLC3A2 in human osteosarcoma (OS) is rarely discussed. Thus, the aim of the present study was to demonstrate the expression of SLC3A2 in human osteosarcoma and reveal its biological function and the underlying mechanisms. RT-PCR, western blot analysis and immunohistochemistry (IHC) were used to assess the expression of SLC3A2 in OS samples and cell lines. Cell cycle, Cell Counting Kit-8 (CCK-8) and colony formation assays were used to test the cell survival capacity. To investigate the potential mechanism by which SLC3A2 regulates OS growth, we used a slide-based antibody array. We demonstrated that SLC3A2 was upregulated in OS cell lines as well as OS tissues. High expression of SLC3A2 was correlated with clinical stage and tumor size in OS. Reduced expression of SLC3A2 inhibited OS cell proliferation through G2/M phase arrest. Most importantly, we found that SLC3A2 may regulate OS growth through the PI3K/Akt signaling pathway. In conclusion, SLC3A2 is upregulated in OS and plays a crucial role in tumor growth. Targeting SLC3A2 may provide a new therapeutic strategy for OS.

Chromobox Homolog 4 is Positively Correlated to Tumor Growth, Survival and Activation of HIF-1α Signaling in Human Osteosarcoma under Normoxic Condition.

Objectives: The clinical significance and tumorigenesis of Chromobox homolog 4 (CBX4) have been reported in hepatocellular carcinoma. The purpose of this study is to confirm the expression, elucidate the biological function and investigate the potential mechanism of CBX4 in osteosarcoma (OS). Methods: The expression of CBX4 in OS samples and cell lines was measured by RT-PCR and western blot test. Cell cycle, CCK8 and colony-forming assays were used to detect changes of cells growth. Cell apoptosis assay was used to measure cell survival capacity. Trans-well assay was used to test the activities of migration and invasion. The expression of genes regulated by CBX4 was detected by qRT-PCT test. Results: The expression of CBX4 was up-regulated in multiple OS cell lines and clinical samples. Overexpression of CBX4 was correlated with advanced clinical stage, high degree of malignancy and low tumor necrosis rate. Moreover, knockdown of CBX4 resulted in significant inhibition of cell growth and cell survival in OS cells under normoxic condition. In addition, we found that knockdown of CBX4 lead to down-regulating of HIF-1α-targeted genes without changing HIF-1α expression itself. Conclusion: Taken together, CBX4 is up-regulated and has a pro-tumor effect in OS with an activation of HIF-1α signaling pathway under normoxic condition. Therefore, targeting CBX4 may provide a new therapeutic method for OS.

Minichromosome maintenance protein 2 and 3 promote osteosarcoma progression via DHX9 and predict poor patient prognosis

A label free quantitative proteomic approach (SWATH™ experiment) was performed to identify tumor-associated nuclear proteins that are differentially expressed between osteosarcoma cells and osteoblast cells. By functional screening, minichromosome maintenance protein 2 (MCM2) and minichromosome maintenance protein 3 (MCM3) were found to be related to osteosarcoma cell growth. Here, we show that knockdown of MCM2 or MCM3 inhibits osteosarcoma growth in vitro and in vivo. In co-immunoprecipitation and co-localization experiments, MCM2 and MCM3 were found to interact with DExH-box helicase 9 (DHX9) in osteosarcoma cells. A rescue study showed that the decreased growth of osteosarcoma cells by MCM2 or MCM3 knockdown was reversed by DHX9 overexpression, indicating that MCM2 and MCM3 activity was DHX9-dependent. In addition, the depletion of DHX9 hindered osteosarcoma cell proliferation. Notably, MCM2 and MCM3 expression levels were positively correlated with the DHX9 expression level in tumor samples and were associated with a poor prognosis in patients with osteosarcoma. Taken together, these results suggest that the MCM2/MCM3–DHX9 axis has an important role in osteosarcoma progression.

The Overexpression of CARM1 Promotes Human Osteosarcoma Cell Proliferation through the pGSK3β/β-Catenin/cyclinD1 Signaling Pathway

Osteosarcoma (OS) is a kind of malignant bone tumor that occurs frequently in the region surrounding the knee joint and poses a threat to the health of teenagers. Since the application of chemotherapy to treat OS, 5-year survival rate in patients has improved from 10% to 70%, but patient survival has not changed over the past four decades. Coactivator-associated arginine methyltransferase 1 (CARM1) is a member of the PRMT protein family; it acts as an oncogene in many cancers, but its function in OS is still unknown. In this study, we found that CARM1 is overexpressed in OS and its expression is correlated with the Enneking stage. CCK-8 and colony forming assays showed that proliferation in OS cell lines was downregulated when siRNA was used to knockdown CARM1 expression. The cell cycle was inhibited in S phase after si-CARM1 transfection in OS cell lines. An antibody array indicated that Erk1/2 (Thr202/Tyr204), PARS40 (Thr246), and GSK3β (Ser9) expression are affected by CARM1, and western blotting verified that CARM1 promotes OS cell proliferation via pGSK3β/β-catenin/cyclinD1 signaling. Accordingly, CARM1 is a crucial gene in OS and is a potential new treatment target.