Clinical significance and immune landscape of a novel ferroptosis-related prognosis signature in osteosarcoma.

BACKGROUND: Osteosarcoma is a malignant tumor that usually occurs in adolescents aged 10-20 years and is associated with poor prognosis. Ferroptosis is an iron-dependent cell death mechanism that plays a vital role in cancer. METHODS: Osteosarcoma transcriptome data were downloaded from the public database TARGET and from previous studies. A prognostic risk score signature was constructed using bioinformatics analysis, and its efficacy was determined by analyzing typical clinical features. The prognostic signature was then validated with external data. Differences in immune cell infiltration between high- and low-risk groups were analyzed. The potential of the prognostic risk signature as a predictor of immunotherapy response was evaluated using the GSE35640 (melanoma) dataset. Five key genes expression were measured by real-time PCR and western blot in human normal osteoblasts and osteosarcoma cells. Moreover, malignant biological behaviors of osteosarcoma cells were tested by modulating gene expression level. RESULTS: We obtained 268 ferroptosis-related genes from the online database FerrDb and published articles. Transcriptome data and clinical information of 88 samples in the TARGET database were used to classify genes into two categories using clustering analysis, and significant differences in survival status were identified. Differential ferroptosis-related genes were screened, and functional enrichment showed that they were associated with HIF-1, T cells, IL17, and other inflammatory signaling pathways. Prognostic factors were identified by univariate Cox regression and LASSO analysis, and a 5-factor prognostic risk score signature was constructed, which was also applicable for external data validation. Experimental validation indicated that the mRNA and protein expression level of MAP3K5, LURAP1L, HMOX1 and BNIP3 decreased significantly, though meanwhile MUC1 increased in MG-63 and SAOS-2 cells compared with hFOB1.19 cells. Cell proliferation and migration ability of SAOS-2 were affected based on alterations of signature genes. CONCLUSIONS: Significant differences in immune cell infiltration between high- and low-risk groups indicated that the five ferroptosis-related prognostic signature was constructed and could be used to predict the response to immunotherapy in osteosarcoma.

Corrigendum: Identification of a Two-Gene (PML-EPB41) Signature With Independent Prognostic Value in Osteosarcoma.

[This corrects the article DOI: 10.3389/fonc.2019.01578.].

Construction of Gene Modules and Analysis of Prognostic Biomarkers for Cervical Cancer by Weighted Gene Co-Expression Network Analysis.

BACKGROUND: Despite advances in the understanding of neoplasm, patients with cervical cancer still have a poor prognosis. Identifying prognostic markers of cervical cancer may enable early detection of recurrence and more effective treatment. METHODS: Gene expression profiling data were acquired from the Gene Expression Omnibus database. After data normalization, genes with large variation were screened out. Next, we built co-expression modules by using weighted gene co-expression network analysis to investigate the relationship between the modules and clinical traits related to cervical cancer progression. Functional enrichment analysis was also applied on these co-expressed genes. We integrated the genes into a human protein-protein interaction (PPI) network to expand seed genes and build a co-expression network. For further analysis of the dataset, the Cancer Genome Atlas (TCGA) database was used to identify seed genes and their correlation to cervical cancer prognosis. Verification was further conducted by qPCR and the Human Protein Atlas (HPA) database to measure the expression of hub genes. RESULTS: Using WGCNA, we identified 25 co-expression modules from 10,016 genes in 128 human cervical cancer samples. After functional enrichment analysis, the magenta, brown, and darkred modules were selected as the three most correlated modules for cancer progression. Additionally, seed genes in the three modules were combined with a PPI network to identify 31 tumor-specific genes. Hierarchical clustering and Gepia results indicated that the expression quantity of hub genes NDC80, TIPIN, MCM3, MCM6, POLA1, and PRC1 may determine the prognosis of cervical cancer. Finally, TIPIN and POLA1 were further filtered by a LASSO model. In addition, their expression was identified by immunohistochemistry in HPA database as well as a biological experiment. CONCLUSION: Our research provides a co-expression network of gene modules and identifies TIPIN and POLA1 as stable potential prognostic biomarkers for cervical cancer.

Paeoniflorin Attenuates Dexamethasone-Induced Apoptosis of Osteoblast Cells and Promotes Bone Formation via Regulating AKT/mTOR/Autophagy Signaling Pathway.

Paeoniflorin, a natural product derived from Paeonia lactiflora, possesses diverse pharmacological activities such as anti-inflammatory, antitumor, and antidiabetic effects. It has been reported for promoting osteoblastogenesis and inhibiting osteoclastogenesis. This study investigates the therapeutic effects of paeoniflorin in glucocorticoid-induced osteoporosis (GIOP) in vitro and in vivo. MC3T3-E1 cells were incubated with dexamethasone (DEX; 200 µM) and/or paeoniflorin (10 µM), followed by the investigation of cell proliferation, differentiation, mineralization, apoptosis, and autophagy. The AKT activator SC79 was used for evaluating the involvement of the AKT/mTOR signaling pathway. After DEX pretreatments, paeoniflorin promoted osteoblast differentiation and mineralization characterized by increase in Runx2, ALP, beclin-1, and LC3-II/LC3-I ratio levels and a decrease in apoptosis. The autophagy-promoting effects of paeoniflorin were reversed by SC79. C57BL/6 mice were given DEX (1 mg/kg) once daily and paeoniflorin (15 mg/kg) 48 hours for a total of 8 weeks followed by the investigation of histological changes, the trabecular bone microarchitecture, and the levels of bone turnover markers. The results showed that paeoniflorin increased alkaline phosphatase (ALP) activity and upregulated the expression of osteocalcin and beclin-1 but reduced the levels of Bax and C-terminal telopeptide of type I collagen (CTX-1). Thus, paeoniflorin may alleviate DEX-induced osteoporosis by promoting osteogenic differentiation and autophagy via inhibition of the AKT/mTOR signaling pathway.

Predictive value of serum VEGF, IL-1 and TNF-α in the treatment of thromboangiitis obliterans by revascularization.

Effect of revascularization in the treatment of thromboangiitis obliterans (TAO) and the predictive value of serum vascular endothelial growth factor (VEGF), interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α) of risk factors of amputation were investigated. From April 2012 to August 2015, a total of 117 patients with TAO admitted to the First Hospital of Lanzhou University were selected. Patients treated with revascularization combined with prostaglandin sodium and cilostazol were enrolled in group A (67 patients), and patients treated with sodium and cilostazol were enrolled in group B (50 patients). The clinical efficacy was evaluated by calculating the intermittent claudication distance and the ankle brachial index (ABI) of patients. The occurrence probability of nausea and vomiting, skin pruritus, abdominal pain, coagulation abnormalities and amputation were recorded. The concentration of serum VEGF, IL-1 and TNF-α were measured using enzyme-linked immunosorbent assay (ELISA). After treatment, the intermittent claudication distance, ABI and efficiency of group A was markedly higher than that of group B (P<0.05). After treatment, serum VEGF concentration in group A was clearly higher than that in group B (P<0.05), and IL-1 and TNF-α levels were much lower than those in group B (P<0.05). The amputation rate in group A was significantly lower than that in group B (P<0.05). Patients with amputation in both groups were enrolled in the study group (24 cases), and those without amputation were included in the control group (93 cases). The serum VEGF concentration in the study group before treatment was significantly lower than that in the control group (P<0.05), while IL-1 and TNF-α levels were significantly higher than those of the control group (P<0.05). In conclusion, pretreatment serum VEGF, IL-1 and TNF-α had a positive diagnostic value for poor prognosis of patients with amputation, and low concentration of VEGF and higher concentration of IL-1 and TNF-α are the risk factors for amputations in patients with TAO.

Emerging Roles and Potential Biological Value of CircRNA in Osteosarcoma.

Circular RNAs (circRNAs) are endogenous noncoding RNAs that are widely found in eukaryotic cells. They have been found to play a vital biological role in the development of human diseases. At present, circRNAs have been involved in the pathogenesis, diagnosis, and targeted treatment of multiple tumors. This article reviews the research progress of circRNAs in osteosarcoma (OSA) in recent years. The potential connection between circRNAs and OSA cell proliferation, apoptosis, metastasis, and chemotherapy sensitivity or resistance, as well as clinical values, is described in this review. Their categories and functions are generally summarized to facilitate a better understanding of OSA pathogenesis, and findings suggest novel circRNA-based methods may be used to investigate OSA and provide an outlook for viable biomarkers and therapeutic targets.

MicroRNA­93 regulates angiogenesis in peripheral arterial disease by targeting CDKN1A.

MicroRNAs (miRNAs) are considered to be critical mediators of gene expression with respect to tumor progression, although their role in ischemia­induced angiogenesis is poorly characterized, including in peripheral arterial disease (PAD). Furthermore, the underlying mechanism of action of specific miRNAs in PAD remains unknown. Reverse transcription­quantitative polymerase chain reaction analysis revealed that microRNA­93 (miR­93) was significantly upregulated in patients with PAD and in the EA.hy926 endothelial cells in response to hypoxia. Additionally, miRNA (miR)­93 promoted angiogenesis by enhancing proliferation, migration and tube formation. Cyclin dependent kinase inhibitor 1A (CDKN1A), verified as a potential target gene of miR­93, was inhibited by overexpressed miR­93 at the protein and mRNA expression levels. Furthermore, a hind­limb ischemia model served to evaluate the role of miR­93 in angiogenesis in vivo, and the results demonstrated that miR­93 overexpression enhanced capillary density and perfusion recovery from hind­limb ischemia. Taken together, miR­93 was indicated to be a promising target for pharmacological regulation to promote angiogenesis, and the miR­93/CDKN1A pathway may function as a novel therapeutic approach in PAD.

Arbutin promotes MC3T3­E1 mouse osteoblast precursor cell proliferation and differentiation via the Wnt/ß­catenin signaling pathway.

Arbutin is a natural compound extracted from various plants, including bearberry leaves, that exerts multiple effects including skin whitening, anti­inflammatory and oxidative stress­protective properties. However, the effects of arbutin on osteoblasts remain unknown. The aim of the present study was to investigate the function and the mechanisms of arbutin on the proliferation and differentiation of MC3T3­E1 mouse osteoblast precursor cells in vitro. The proliferation of MC3T3­E1 cells treated with arbutin was assessed using a Cell Counting Kit­8 assay and a 5­ethynyl­2'­deoxyuridine labeling assay. Additionally, cell cycle and apoptosis were examined using flow cytometry analysis. The effects of arbutin on osteoblast differentiation were investigated using alkaline phosphatase (ALP) staining and by examining the mRNA expression levels of collagen type I α1 chain (COL1A1), bone γ­carboxyglutamate protein (BGLAP) and Sp7 transcription factor (SP7). To further investigate the molecular mechanism underlying arbutin function in promoting osteogenesis, the mRNA and protein expression levels of runt­related transcription factor 2 (RUNX2) and ß­catenin were analyzed by reverse transcription­quantitative polymerase chain reaction and western blotting. Arbutin significantly promoted MC3T3­E1 cell proliferation and increased the ratio of cells in S­phase. Treatment with arbutin increased ALP activity and the mRNA expression levels of COL1A1, BGLAP and SP7 in MC3T3­E1 cells. Furthermore, the protein and the mRNA expression levels of RUNX2 and ß­catenin increased significantly following treatment with arbutin. Collectively, the present findings suggested that arbutin was able to promote proliferation and differentiation of MC3T3­E1 cells via the Wnt/ß­catenin signaling pathway.

Identification of a Two-Gene (PML-EPB41) Signature With Independent Prognostic Value in Osteosarcoma.

Background: Osteosarcoma (OSA) is the most prevalent form of malignant bone cancer and it occurs predominantly in children and adolescents. OSA is associated with a poor prognosis and highest cause of cancer-related death. However, there are a few biomarkers that can serve as reasonable assessments of prognosis. Methods: Gene expression profiling data were downloaded from dataset GSE39058 and GSE21257 from the Gene Expression Omnibus database as well as TARGET database. Bioinformatic analysis with data integration was conducted to discover the significant biomarkers for predicting prognosis. Verification was conducted by qPCR and western blot to measure the expression of genes. Results: 733 seed genes were selected by combining the results of the expression profiling data with hub nodes in a human protein-protein interaction network with their gene functional enrichment categories identified. Following by Cox proportional risk regression modeling, a 2-gene (PML-EPB41) signature was developed for prognostic prediction of patients with OSA. Patients in the high-risk group had significantly poorer survival outcomes than in the low-risk group. Finally, the signature was validated and analyzed by the external dataset along with Kaplan-Meier survival analysis as well as biological experiment. A molecular gene model was built to serve as an innovative predictor of prognosis for patients with OSA. Conclusion: Our findings define novel biomarkers for OSA prognosis, which will possibly aid in the discovery of novel therapeutic targets with clinical applications.

Leonurine hydrochloride promotes osteogenic differentiation and increases osteoblastic bone formation in ovariectomized mice by Wnt/ß-catenin pathway.

Leonurine hydrochloride (LH) is a synthetic chemical compound derived from leonurine that can be extracted from Leonurus sibiricus and possesses antioxidant, anti-apoptosis, and neuroprotective activities. In previous studies, LH has been demonstrated to attenuate osteoclast activity and prevent bone loss. However, it is unknown whether LH accelerates bone formation and promotes osteogenic differentiation. We systematically examined the effects of LH on ovariectomized-induced osteoporotic mice and the MC3T3-E1 osteoblastic cell line. The results revealed that LH enhanced differentiation of MC3T3-E1 cells, with a dose-dependent increase in alkaline phosphatase (ALP) activity. Moreover, LH upregulated osteogenesis-related gene expression, including osterix, alpha 1 type 1 collagen, runt-related transcription factor 2 (Runx2) and ALP, as shown by quantitative reverse transcription-polymerase chain reaction analysis. At the same time, elevated expression of low-density lipoprotein receptor-related protein 5 and ß-catenin mRNA was detected in the Wnt/ß-catenin pathway. A western blot analysis revealed that LH dose-dependently increased the expression of Runx2 and ß-catenin, and promoted phosphorylation of glycogen synthase kinase-3ß in vitro. The in vivo results showed that administering LH (15 mg/kg/d) for 8 weeks alleviated destruction of the trabecular microstructure caused by osteoporosis. LH increased the bone mineral density and trabecular number, decreased trabecular separation according to a micro-computed tomography scan. In addition, LH enhanced the expression of ß-catenin and Runx2 in vivo. In conclusion, LH promoted osteogenic differentiation and bone formation in vivo and in vitro, which alleviated osteoporosis through activation of the Wnt/ß-catenin pathway.

Shikonin stimulates MC3T3-E1 cell proliferation and differentiation via the BMP-2/Smad5 signal transduction pathway.

Shikonin, the predominant naphthoquinone pigment isolated from the Chinese plant Lithospermum erythrorhizon, is anti­inflammatory, antiviral and exerts anticancer effects, amongst other biological activities. However, it is unknown whether shikonin affects bone formation. In the present study, the role of shikonin on cell proliferation was assessed via MTT assay, and shikonin was identified to markedly promote cell growth in a time­ and dose­dependent manner in the MC3T3­E1 cell line. In addition, flow cytometric analysis was performed to evaluate the effect of shikonin on the cell cycle, and it was observed that shikonin markedly increased the percentage of S­phase MC3T3­E1 cells to accelerate the G1/S transition. To investigate the potential molecular mechanism by which shikonin enhances bone formation, the changes in bone morphogenic protein­2 (BMP­2), SMAD family member 5 (Smad5), runt related transcription factor 2 (Runx2), alkaline phosphatase (ALP) and osteocalcin (OC) expression levels induced by shikonin were investigated using western blot analysis and quantitative polymerase chain reaction. The results indicated that shikonin increased the BMP­2 and Smad5 mRNA levels, and upregulated Smad5 and Runx2 protein expression levels to promote osteoblast differentiation. Furthermore, ALP staining was performed, and revealed that shikonin enhanced ALP activity. These results indicate that shikonin promotes cell proliferation and differentiation of MC3T3-E1 cells via the BMP-2/Smad5 signaling pathway.

TRIM29 Overexpression Promotes Proliferation and Survival of Bladder Cancer Cells through NF-κB Signaling.

PURPOSE: TRIM29 overexpression has been reported in several human malignancies and showed correlation with cancer cell malignancy. The aim of the current study is to examine its clinical significance and biological roles in human bladder cancer tissues and cell lines. MATERIALS AND METHODS: A total of 102 cases of bladder cancer tissues were examined for TRIM29 expression by immunohistochemistry. siRNA and plasmid transfection were performed in 5637 and BIU-87 cell lines. Cell Counting Kit-8, flow cytometry, western blot, and real-time polymerase chain reaction were performed to examine its biological roles and mechanism in bladder cancer cells. RESULTS: We found that TRIM29 overexpression showed correlation with invading depth (p=0.0087). Knockdown of TRIM29 expression in bladder cancer cell line 5637 inhibited cell growth rate and cell cycle transition while its overexpression in BIU-87 cells accelerated cell proliferation and cell cycle progression. TRIM29 overexpression also inhibited cell apoptosis induced by cisplatin. In addition, we demonstrated that TRIM29 depletion decreased while its overexpression led to upregulated expression of cyclin D1, cyclin E, and Bcl-2. We also showed that TRIM29 knockdown inhibited protein kinase C (PKC) and nuclear factor κB (NF-κB) signaling while its overexpression stimulated the PKC and NF-κB pathways. BAY 11-7082 (NF-κB inhibitor) partly attenuated the effect of TRIM29 on expression of cyclin and Bcl-2. Treatment with PKC inhibitor staurosporine resulted in ameliorated TRIM29 induced activation of NF-κB. CONCLUSION: The current study demonstrated that TRIM29 upregulates cyclin and Bcl family proteins level to facilitate malignant cell growth and inhibit drug-induced apoptosis in bladder cancer, possibly through PKC-NF-κB signaling pathways.