OBJECTIVE: To investigate the prognostic value and mechanism of long non-coding RNA DLEU1(LncRNA DLEU1) in osteosarcoma. METHODS: The tissue samples and clinical data of 86 patients with osteosarcoma treated by orthopaedic surgery in our hospital from January 2012 to December 2014 were retrospectively collected. The expression of LncRNA DLEU1 in pathological tissues was detected by qRT-PCR, then the patients were divided into high and low expression of LncRNA DLEU1 groups. Osteosarcoma cell line HOS was divided into two groups, down-regulated expression group (si-DLEU1 group) and negative control group (si-NC group). LncRNA DLEU1 siRNA and negative control sequence were transfected by Lipofectamine 3000. Chi-square test was used to analyze the relationship between the expression of LncRNA DLEU1 and the clinicopathological factors of osteosarcoma. Kaplan-Meier method was used to compare the difference of the overall survival rate of osteosarcoma patients between the high and low expression groups of LncRNA DLEU1. The risk factors affecting the overall survival rate of osteosarcoma were analyzed by single factor and multifactor analysis. The number of invasive cells in the two groups was determined and compared by Transwell assay. RESULTS: The expression of LncRNA DLEU1 in osteosarcoma tissue was higher than that in adjacent tissues (P<0.001). The expression of LncRNA DLEU1 in human osteosarcoma cell lines (MG-63, U-2 OS, and HOS) was significantly higher than that in human osteoblast line hFOB 1.19 (P<0.001). The expression of LncRNA DLEU1 was significantly correlated with Enneking stage (P<0.001), distant metastasis (P=0.016), and histological grade (P=0.028). The 1-year overall survival rate of the LncRNA DLEU1 high expression group was significantly higher than that of the low expression group (90.7% vs 60.5%, P<0.001). The 5-year overall survival rate of the LncRNA DLEU1 high expression group was significantly higher than that of the low expression group (32.6% vs 11.6%, P<0.001). Univariate analysis showed that Enneking stage (P<0.001), tumor size (P=0.043), distant metastasis (P<0.001), histological grade (P<0.001), and expression of LncRNA DLEU1 (P<0.001) were risk factors for overall survival of osteosarcoma patients. Multivariate analysis showed that high expression of LncRNA DLEU1 [HR=1.948, 95% CI(1.141, 3.641), P=0.012] and distant metastasis[HR=4.108, 95% CI(2.169, 7.780), P<0.001] were independent risk factors for overall survival of osteosarcoma patients. The number of invasive cells in si-DLEU1 group was significantly lesser than that in si-NC group(139±13 vs 357±31, P<0.001). CONCLUSION High expression of LncRNA DLEU1 is a molecular marker affecting the prognosis of osteosarcoma patients. Downregulation of LncRNA DLEU1 can inhibit the invasion of osteosarcoma cells.
Humans ; Prognosis ; RNA, Long Noncoding/metabolism* ; Retrospective Studies ; Cell Proliferation/genetics* ; Cell Line, Tumor ; Osteosarcoma/genetics* ; Bone Neoplasms/pathology*

Humans ; MicroRNAs/genetics* ; Cell Line, Tumor ; Osteosarcoma/pathology* ; Neoplasm Invasiveness ; Cell Proliferation ; Bone Neoplasms/pathology* ; Cell Movement ; Neoplasm Metastasis

OBJECTIVE: To explore the effect of miR-143 regulating matrix metalloproteinase(MMP)-13 expression on migration and invasion of osteosarcoma cells. METHODS: The mouse osteosarcoma cell line 143B cells were cultured in 96-well plates, and blank group, negative group, positive group, and intervention group were set up. Then, the blank group did no treatment 50 μg miR-143 mimic was added to positive group, negative group added equal mimic NC (control sequence of miR-143 mimic), the intervention group was added 50 μg miR-143 mimic and 10 μg MMP-13 protein, all groups continued to culture for 3 to 6 hours, and finally the serum was aspirated to treat for half an hour. The protein expressions of miR-143 and MMP-13 in each group were measured by fluorescence quantitative PCR experiment and Western blot experiment, respectively, and the invasion and migration abilities of cells were measured by Transwell and scratch experiments. RESULTS: The expression of MMP-13 protein in the positive group and the intervention group was significantly lower than that in the blank group, and the positive group was lower than the intervention group (P<0.05);The mean numbers of invasive cells in blank group, negative group, positive group and intervention group were (1 000.01±44.77), (959.25±46.32), (245.04±4.33), (634.06±33.78) cells/field, respectively;the scratch healing rate of the positive group and the intervention group was significantly lower than that of the blank group, and the positive group was lower than the intervention group (P<0.05). CONCLUSION MMP-13 is a target of miR-143, which can reduce the migration and invasion ability of osteosarcoma cells by inhibiting the expression of MMP-13.
Osteosarcoma/pathology* ; MicroRNAs/genetics* ; Matrix Metalloproteinase 13/genetics* ; Neoplasm Invasiveness ; Animals ; Mice ; Cell Line, Tumor ; Cell Movement

Objective To explore the cell subsets and characteristics related to the prognosis of osteosarcoma by analyzing the cellular composition of tumor tissue samples from different osteosarcoma patients.Methods The single-cell sequencing data and bulk sequencing data of different osteosarcoma patients were downloaded.We extracted the information of cell samples for dimensionality reduction,annotation,and cell function analysis,so as to identify the cell subsets and clarify the cell characteristics related to the prognosis of osteosarcoma.The development trajectory of macrophages with prognostic significance was analyzed,and the prognostic model of osteosarcoma was established based on the differentially expressed genes of macrophage differentiation.Results The cellular composition presented heterogeneity in the patients with osteosarcoma.The infiltration of mononuclear phagocytes in osteosarcoma had prognostic significance(P=0.003).Four macrophage subsets were associated with prognosis,and their signature transcription factors included RUNX3(+),ETS1(+),HOXD11(+),ZNF281(+),and PRRX1(+).Prog_Macro2 and Prog_Macro4 were located at the end of the developmental trajectory,and the prognostic ability of macrophage subsets increased with the progression of osteosarcoma.The prognostic model established based on the differentially expressed genes involved in macrophage differentiation can distinguish the survival rate of osteosarcoma patients with different risks(P<0.001).Conclusion Macrophage subsets are closely related to the prognosis of osteosarcoma and can be used as the key target cells for the immunotherapy of osteosarcoma.
Humans ; Prognosis ; Osteosarcoma/genetics* ; Immunotherapy ; Macrophages ; Transcription Factors ; Bone Neoplasms/genetics* ; Homeodomain Proteins ; Repressor Proteins

OBJECTIVE: To investigate the inhibitory effect of miR-125b-5p on proliferation and migration of osteosarcoma and the role of RAB3D in mediating this effect. METHODS: The expression level of miR-125b-5p was detected by qRT-PCR in a normal bone cell line (hFOB1.19) and in two osteosarcoma OS cell lines (MG63 and HOS). A miR-125b-5p mimic or inhibitor was transfected in the osteosarcoma cell lines via liposome and the changes in cell proliferation and migration were detected with EDU and Transwell experiments. Bioinformatic analysis was conducted for predicting the target gene of miR-125b-5p, and the expression level of RAB3D in hFOB1.19, MG63, and HOS cells was detected by Western blotting. In the two osteosarcoma cell lines transfected with miR-125b-5p mimic or inhibitor, the expression levels of RAB3D mRNA and protein in osteosarcoma cells were examined with qRT-PCR and Western blotting. The effects of RAB3D overexpression, RAB3D knockdown, or overexpression of both miR-125b-5p and RAB3D on the proliferation and migration of cells were assessed using EDU and Transwell experiments. RESULTS: The two osteosarcoma cell lines had significantly lower expression levels of miR-125b-5p (P < 0.05). Bioinformatic analysis predicted that RAB3D was a possible target gene regulated by miR-125b-5p. In osteosarcoma cells, overexpression of miR-125b-5p significantly lowered the expression of RAB3D protein (P < 0.05); inhibiting miR-125b-5p expression significantly decreased RAB3D expression only at the protein level (P < 0.05) without obviously affecting its mRNA level. Modulation of miR-125b-5p and RAB3D levels produced opposite effects on proliferation and migration of osteosarcoma cells, and in cells with overexpression of both miR-125b-5p and RAB3D, the effect of RAB3D on cell proliferation and migration was blocked by miR-125b-5p overexpression (P < 0.05). CONCLUSION Overexpression of miR-125b-5p inhibits the proliferation and migration of osteosarcoma cells by regulating the expression of RAB3D at the post-transcriptional level.
Humans ; Bone Neoplasms/genetics* ; Cell Proliferation ; MicroRNAs/genetics* ; Osteosarcoma/genetics* ; rab3 GTP-Binding Proteins/genetics* ; RNA, Messenger

Objective: To explore the effects of circTNPO1 on the proliferation and metastasis of osteosarcoma (OS) by sponging miR-338-3p. Methods: The expression of circTNPO1 on osteoblasts and multiple OS cell lines were detected by qRT-PCR. CircTNPO1 stable knockdown 143B cell line was constructed by sh-circTNPO1. Cell count kit 8 (CCK-8) assay and wound healing assay were applied to evaluate the proliferation and metastasis of this cell. Luciferase reporter assay was used to explore the binding between circTNPO1 and miR-338-3p. In xenograft tumor model, miR-338-3p inhibitor or its control was injected into the circTNPO1 knockdown tumors. The weight and size of the tumors were evaluated and Ki-67 expression was detected by immunohistochemistry. Results: The RNA expression of circTNPO1 in OS cell lines U2OS, HOS, MG63, 143B, ZOS and ZOSM were 2.73±0.27, 3.18±0.54, 4.33±0.52, 5.75±0.65, 4.50±0.49 and 3.96±0.35, respectively, higher than 1.00±0.09 in hFOB1.19 (P<0.001). CCK-8 assay revealed that after 48 h and 72 h, the absorbance of sh-circTNPO1 #1 was 0.81±0.05 and 1.09±0.06, while sh-circTNPO1 #2 143B cells was 0.84±0.04 and 1.2±0.04, which were sharply reduced compared with the control (1.00±0.06 and 1.49±0.06, P<0.001); after 48 h and 72 h, the absorbance of 143B cells transfected with circTNPO1 #1 and miR-338-3p (0.92±0.06 and 1.32±0.07) were higher than those of cells transfected with sh-circTNPO1 cells and miR NC (0.92±0.06 and 1.32±0.07, P<0.050). Wound healing assay demonstrated that the 24 hour-migration rates of sh-circTNPO1 #1 and sh-circTNPO1 #2 cells were (24.43±2.15)% and (39.70±4.20)% respectively, which were significantly lower than that of the control [(56.51±3.27)%, P<0.010]; the migration rates of sh-circTNPO1 #1+ miR NC and sh-circTNPO1 #1+ miR-338-3p inhibitor were (26.70±2.21)% and (46.10±5.71)%, with a significant difference (P<0.005). In xenograft tumor model, the weight and size of tumors in control, sh-circTNPO1 #1+ miR NC and sh-circTNPO1 #1+ miR-338-3p inhibitor mice were (458.80±158.10) mg, (262.50±82.09) mg, (395.40±137.60) mg and (593.00±228.40) mm(2,) (203.30±144.20) mm(2,) (488.60±208.60) mm(2,) respectively. Compared with control, sh-circTNPO1 tumors were significantly smaller (P<0.01). Injection with miR-338-3p inhibitor significantly reversed both the weight and size of tumors (P<0.05). Conclusion: CircTNPO1 promotes the proliferation and metastasis of OS by sponging miR-338-3p, which could be a new target for OS treatments.
Animals ; Bone Neoplasms/pathology* ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation/genetics* ; Gene Expression Regulation, Neoplastic ; Humans ; Ki-67 Antigen/metabolism* ; Mice ; MicroRNAs/metabolism* ; Osteosarcoma/secondary* ; RNA, Circular/metabolism* ; Sincalide/metabolism*

OBJECTIVE: To screen for aberrantly expressed genes in osteosarcoma cells and investigate the role of RHPN2 in regulating the proliferation, apoptosis, migration and tumorigenic abilities of osteosarcoma cells. METHODS: We used GEO2R to analyze the differential gene expression profile between osteosarcoma cells and normal cells in the GSE70414 dataset. RTqPCR and Western blotting were performed to detect RHPN2 expression in osteosarcoma cell lines MG-63, 143B and SAOS2. Two RHPN2-shRNA and a control NC-shRNA were designed to silence the expression of RHPN2 in 143B cells, and CCK8 assay, colony-forming assay, annexin V-FITC/PI staining and scratch assays were carried out to examine the changes in proliferation, apoptosis and migration of the cells. We also established nude mouse models bearing osteosarcoma xenografts derived 143B cells and RHPN2-shRNA-transfected 143B cells, and assessed the effect of RHPN2 silencing on osteosarcoma cell tumorigenesis using HE staining. Kaplan-Meier survival curves were used to analyze the correlation between RHPN2 expression and survival outcomes of patients with osteosarcoma. RESULTS: RHPN2 expression was significantly upregulated in osteosarcoma cell lines MG-63, 143B and SAOS2 (P < 0.01). Silencing of RHPN2 significantly inhibited the proliferation and migration of 143B cells in vitro, promoted cell apoptosis (P < 0.01), and suppressed tumorigenic capacity of the cells in nude mice. A high expression of RHPN2 was significantly correlated with a poor prognosis of patients with osteosarcoma (P < 0.05). CONCLUSION RHPN2 is highly expressed in osteosarcoma cells to promote cell proliferation and migration and inhibits cell apoptosis. A high expression of RHPN2 is associated with a poorer prognosis of the patients with osteosarcoma.
Adaptor Proteins, Signal Transducing/metabolism* ; Animals ; Apoptosis ; Bone Neoplasms/metabolism* ; Carcinogenesis ; Cell Line, Tumor ; Cell Movement/physiology* ; Cell Proliferation/physiology* ; Humans ; Immediate-Early Proteins ; Mice ; Mice, Nude ; Osteosarcoma/metabolism* ; RNA, Small Interfering/genetics*

OBJECTIVE: To study the effects and mechanisms of miR-181a-5p on the proliferation, cycle and migration of HOS osteosarcoma cells. METHODS: Real-time quantitative PCR was used to detect the expression of miR-181a-5p and HOXB4 in osteoblast hFOB1.19 cell line and osteosarcoma cell lines (HOS, U2OS, MG63). miR-181a-5p mimics and miR-181a-5p inhibitors were respectively transfected into HOS cells by Lipofectamine 2000, and miR NC group was set as control group. CCK-8 method was used to detect the change in cell proliferation. Flow cytometry was used to detect the changes in cell cycles. Wound healing experiments and Transwell migration experiments were used to detect the changes in cell migration ability. The target gene of miR-181a-5p was predicted by Targetscan website and validated by Dual-luciferase reporter gene system and Western blot. RESULTS: Compared with osteoblast hFOB1.19, miR-181a-5p was low expressed in osteosarcoma cells HOS, U2OS, and MG63(P<0.05), while HOXB4 was high expressed in osteosarcoma cells HOS, U2OS, and MG63(P<0.05). Compared with the miR NC group, over expression of miR-181a-5p inhibited the proliferation and migration of osteosarcoma HOS cells, and the number of cells in S phase decreased(P<0.05). However, knockdown miR-181a-5p promoted the proliferation and migration of osteosarcoma HOS cells, the cells in S phase increased(P<0.05). Bioinformatics prediction and Dual-luciferase reporter gene system validate HOXB4 as a downstream target gene of miR-181a-5p(P<0.05). Western blot showed that miR-181a-5p over expression or knockdown significantly down-regulated or up-regulated HOXB4 expressions in the HOS cells respectively(P<0.05). CONCLUSION miR-181a-5p is down expressed in osteosarcoma cells, and over-expression miR-181a-5p inhibits the proliferation, cell cycle and migration ability of osteosarcoma cells by targeting HOXB4.
Humans ; Apoptosis ; Bone Neoplasms/genetics* ; Cell Line, Tumor ; Cell Proliferation/genetics* ; Homeodomain Proteins/genetics* ; MicroRNAs/metabolism* ; Osteosarcoma/genetics* ; Transcription Factors/genetics*

Objective: To screen the different expressed genes between osteosarcoma and normal osteoblasts, and find the key genes for the occurrence and development of osteosarcoma. Methods: The gene expression dataset GSE33382 of normal osteoblasts and osteosarcoma was obtained from Gene Expression Omnibus (GEO) database. The different expressed genes between normal osteoblasts and osteosarcoma were screened by limma package of R language, and the different expressed genes were analyzed by Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis. The protein interaction network was constructed by the String database, and the network modules in the interaction network were screened by the molecular complex detection (MCODE) plug-in of Cytoscape software. The different expressed genes contained in the first three main modules screened by MCODE were analyzed by gene ontology (GO) using the BiNGO module of Cytoscape software. The MCC algorithm was used to screen the top 10 key genes in the protein interaction network. The gene expression and survival dataset GSE39055 of osteosarcoma was obtained from GEO database, and the survival analysis was performed by Kaplan-Meier method. The data of 48 patients with osteosarcoma treated in the First Affiliated Hospital of Fujian Medical University from January 2005 to December 2015 were selected for verification. The expression of STC2 protein in osteosarcoma was detected by immunohistochemical method, and the survival analysis was carried out combined with the clinical data of the patients. Results: A total of 874 different expressed genes were identified from GSE33382 dataset, including 402 down-regulated genes and 472 up-regulated genes. KEGG enrichment analysis showed that different expressed genes were mainly related to p53 signal pathway, glutathione metabolism, extracellular matrix receptor interaction, cell adhesion molecules, folate tolerance, and cell senescence. The top 10 key genes in the interaction network were GAS6, IL6, RCN1, MXRA8, STC2, EVA1A, PNPLA2, CYR61, SPARCL1 and FSTL3. STC2 was related to the survival rate of patients with osteosarcoma (P<0.05). The results showed that the expression of STC2 protein was related to tumor size and Enneking stage in 48 cases of osteosarcoma. The median survival time of 25 cases with STC2 high expression was 21.4 months, and that of 23 cases with STC2 low expression was 65.4 months. The survival rate of patients with high expression of STC2 was lower than that of patients with low expression of STC2 (P<0.05). Conclusions: Bioinformatics analysis can effectively screen the different expressed genes between osteosarcoma and normal osteoblasts. STC2 is one of the important predictors for the prognosis of osteosarcoma.
Bone Neoplasms/pathology* ; Computational Biology/methods* ; Follistatin-Related Proteins/genetics* ; Gene Expression Profiling/methods* ; Gene Expression Regulation, Neoplastic ; Humans ; Osteosarcoma/pathology*

Objective To screen the potential key genes of osteosarcoma by bioinformatics methods and analyze their immune infiltration patterns. Methods The gene expression profiles GSE16088 and GSE12865 associated with osteosarcoma were obtained from the Gene Expression Omnibus(GEO),and the differentially expressed genes(DEGs)related to osteosarcoma were screened by bioinformatics tools.Gene Ontology(GO)annotation,Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment,and analysis of immune cell infiltration were then carried out for the DEGs.The potential Hub genes of osteosarcoma were identified by protein-protein interaction network,and the expression of Hub genes in osteosarcoma and normal tissue samples was verified via the Cancer Genome Atlas(TCGA). Results A total of 108 DEGs were screened out.GO annotation and KEGG pathway enrichment revealed that the DEGs were mainly involved in integrin binding,extracellular matrix (ECM) structural components,ECM receptor interactions,and phosphatidylinositol 3-kinase/protein kinase B(PI3K/Akt)signaling pathway.Macrophages were the predominant infiltrating immune cells in osteosarcoma.Secreted phosphoprotein 1(SPP1),matrix metallopeptidase 2(MMP2),lysyl oxidase(LOX),collagen type V alpha(II)chain(COL5A2),and melanoma cell adhesion molecule(MCAM)presented differential expression between osteosarcoma and normal tissue samples(all P<0.05). Conclusions SPP1,MMP2,LOX,COL5A2,and MCAM are all up-regulated in osteosarcoma,which may serve as potential biomarkers of osteosarcoma.Macrophages are the key infiltrating immune cells in osteosarcoma,which may provide new perspectives for the treatment of osteosarcoma.
Bone Neoplasms/immunology* ; Computational Biology/methods* ; Gene Expression Profiling/methods* ; Humans ; Osteosarcoma/immunology* ; Phosphatidylinositol 3-Kinases/genetics* ; Tumor-Associated Macrophages/immunology*