Bioinformatics Analysis and Experimental Validation of Differential Genes and Pathways in Bone Nonunions.

Non-union fractures pose a significant clinical challenge, often leading to prolonged pain and disability. Understanding the molecular mechanisms underlying non-union fractures is crucial for developing effective therapeutic interventions. This study integrates bioinformatics analysis and experimental validation to unravel key genes and pathways associated with non-union fractures. We identified differentially expressed genes (DEGs) between non-union and fracture healing tissues using bioinformatics techniques. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were employed to elucidate the biological processes and pathways involved. Common DEGs were identified, and a protein-protein interaction (PPI) network was constructed. Fibronectin-1 (FN1), Thrombospondin-1 (THBS1), and Biglycan (BGN) were pinpointed as critical target genes for non-union fracture treatment. Experimental validation involved alkaline phosphatase (ALP) and Alizarin Red staining to confirm osteogenic differentiation. Our analysis revealed significant alterations in pathways related to cell behavior, tissue regeneration, wound healing, infection, and immune responses in non-union fracture tissues. FN1, THBS1, and BGN were identified as key genes, with their upregulation indicating potential disruptions in the bone remodeling process. Experimental validation confirmed the induction of osteogenic differentiation. The study provides comprehensive insights into the molecular mechanisms of non-union fractures, emphasizing the pivotal roles of FN1, THBS1, and BGN in extracellular matrix dynamics and bone regeneration. The findings highlight potential therapeutic targets and pathways for further investigation. Future research should explore interactions between these genes, validate results using in vivo fracture models, and develop tailored treatment strategies for non-union fractures, promising significant advances in clinical management.

Long noncoding RNA ZEB1-AS1 attenuates ferroptosis of gastric cancer cells through modulating miR-429/BGN axis.

Gastric cancer (GC) is the fifth utmost common malignant cancer type globally, in which ferroptosis acts a critical function in the progress of GC. Long noncoding RNA ZEB1-AS1 has been recognized in numerous cancers, but the role of ZEB1-AS1 in ferroptosis remains obscure. Hence, we investigated the efficacy of ZEB1-AS1 on ferroptosis of GC cells. The cell growth and viability were analyzed via cell counting kit assay and xenograft tumor model in vivo and in vitro, respectively. The RNA and protein expression were measured by qRT-PCR and western blot analysis assay, respectively. The levels of Fe2+ , malondialdehyde (MDA), and lipid reactive oxygen species (ROS) were tested to determine ferroptosis. The erastin and RSL3 were used to induce ferroptosis. The mechanism was analyzed via luciferase reporter gene and RIP assays. The treatment of ferroptosis inducer Erastin and RSL3 suppressed the viability of GC cells and the ZEB1-AS1 overexpression rescued the phenotype in the cells. The levels of Fe2+ , MDA, and ROS were enhanced through the depletion of ZEB1-AS1 in Erastin/RSL3 treated GC cells. ZEB1-AS1 directly sponged miR-429 in GC cells and miR-429 targeted BGN in GC cells, and the inhibition of miR-429 rescued ZEB1-AS1 depletion-inhibited BGN expression. We validated that miR-429 induced and BGN-repressed ferroptosis in cancer cells. The BGN overexpression and miR-429 suppression could reverse the efficacy of ZEB1-AS1 on proliferation and ferroptosis in cancer cells. The expression of ZEB1-AS1 and BGN was enhanced and miR-429 expression was decreased in clinical GC tissues. ZEB1-AS1 attenuated ferroptosis of cancer cells by modulating miR-429/BGN axis.

Enhancement of tenogenic differentiation of rat tendon-derived stem cells by biglycan.

Biglycan (BGN) has been identified as one of the critical components of the tendon-derived stem cells (TDSCs) niche and may be related to tendon formation. However, so far, no study has demonstrated whether the soluble BGN could induce the tenogenic differentiation of TDSCs in vitro. The aim of this study was to investigate the effect of BGN on the tenogenic differentiation of TDSCs. The proliferation and tenogenic differentiation of TDSCs exposed to different concentrations of BGN (0, 50, 100, and 500 ng/ml) were determined by the live/dead cell staining assay, CCK-8 assay, quantitative real-time polymerase chain reaction (qRT-PCR), and western blot analysis. The BGN signaling pathway of TDSCs (with and without 50 ng/ml of BGN) was determined by western blot analysis and qRT-PCR analysis. At a concentration of 50 ng/ml, BGN increased the expression of the tenogenic markers THBS-4 and TNMD at both the messenger RNA (mRNA) and protein levels. Meanwhile, 50 ng/ml of BGN inhibited the expression of the chondrogenic and osteogenic markers SOX9, ACN, and RUNX2 at both the mRNA and protein levels. Moreover, BGN (50 ng/ml) affected the expression of the components of the extracellular matrix of TDSCs. Additionally, BGN activated the Smad1/5/8 pathway as indicated by an increase in phosphorylation and demonstrated by inhibition experiments. Upregulation in the gene expression of BMP-associated receptors (BMPRII, ActR-IIa, and BMPR-Ib) and Smad pathway components (Smad4 and 8) was observed. Taken together, BGN regulates tenogenic differentiation of TDSCs via BMP7/Smad1/5/8 pathway and this regulation may provide a basic insight into treating tendon injury.

Avian genomics lends insights into endocrine function in birds.

The genomics era has brought along the completed sequencing of a large number of bird genomes that cover a broad range of the avian phylogenetic tree (>30 orders), leading to major novel insights into avian biology and evolution. Among recent findings, the discovery that birds lack a large number of protein coding genes that are organized in highly conserved syntenic clusters in other vertebrates is very intriguing, given the physiological importance of many of these genes. A considerable number of them play prominent endocrine roles, suggesting that birds evolved compensatory genetic or physiological mechanisms that allowed them to survive and thrive in spite of these losses. While further studies are needed to establish the exact extent of avian gene losses, these findings point to birds as potentially highly relevant model organisms for exploring the genetic basis and possible therapeutic approaches for a wide range of endocrine functions and disorders.

Integrative genomics unveils basement membrane-related diagnostic markers and therapeutic targets in esophageal squamous cell carcinoma.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is often diagnosed at advanced stages due to the inherent limitations of current screening methodologies. Central to evaluating tumor invasion and prognostic assessment in ESCC is the integrity of the basement membrane (BM). However, current research on the implications of BM-related genes (BMRGs) in diagnosing ESCC remains sparse. METHODS: We performed a comprehensive analysis using single-cell RNA-sequencing (scRNA-seq) data from the Gene Expression Omnibus (GEO) database, alongside gene expression profiles acquired from GEO and The Cancer Genome Atlas (TCGA) databases. This identified differentially expressed BMRGs in ESCC. Employing LASSO, RF, and SVM-RFE, we selected potential BM biomarkers and crafted a diagnostic nomogram for ESCC, validated by ROC curves and AUC values. We also explored immune infiltration and biological mechanisms through consensus clustering and GSVA, and utilized single cell trajectory analysis and GSCALite to study gene distributions and pathways. In vitro experiments further elucidated the role of these genes in ESCC carcinogenesis. RESULTS: Here, we discovered that ESCC cell types exhibited markedly elevated BM-related scores. Our analysis pinpointed seven BM genes upregulated and linked to immune infiltration, showcasing unique gene expression profiles and varying immune cell densities across the BM-related subtypes. Furthermore, a robust positive correlation was observed between these genes expression and EMT activity. The knockdown of BGN significantly suppressed cell proliferation, migration, invasion, while also augmenting cell viability following chemotherapy drug treatment. CONCLUSION: Our study identified seven key BMRGs (BGN, LAMB3, SPARC, MMP1, LUM, COL4A1, and NELL2) and established a diagnostic nomogram for ESCC. Of noteworthy significance is the discovery of BGN as a promising drug target, indicating a novel strategy for future clinical combination therapies in ESCC.

Assessment of glycosaminoglycan content in bone using Raman spectroscopy.

Glycosaminoglycans (GAGs) are responsible for preserving bone tissue toughness as well as regulating collagen formation and mineralization in the extracellular matrix. However, current methods for characterization of GAGs in bone are destructive, thus unable to capture in situ changes or differences in GAGs between experimental groups. As an alternative, Raman spectroscopy is a non-destructive method and can detect concurrent changes in GAGs and other bone constituents. In this study, we hypothesized that the two most prominent Raman peaks of sulfated GAGs (at ~1066 cm-1 and at ~1378 cm-1) could be used to detect differences in GAGs content of bone. To test this hypothesis, three experimental models were utilized: an in vitro model (enzymatic removal of GAGs from human cadaver bone), an ex vivo mouse model (biglycan KO vs. WT), and an ex vivo aging model (comparing cadaveric bone samples from young and old donors). All Raman measurements were compared to Alcian blue measurements to confirm the validity of Raman spectroscopy in detecting GAGs changes in bone. Irrespective of different models, it was found that the ~1378 cm-1 peak in Raman spectra of bone was uniquely sensitive to changes of GAGs content in bone when normalized with respect to the phosphate phase (~960 cm-1); i.e., 1378 cm-1/960 cm-1 (peak intensity ratio) or 1370-1385 cm-1/930-980 cm-1 (integrated peak area ratio). In contrast, the 1070 cm-1 peak, which includes another major peak of GAGs (1066 cm-1), seemed to be compromised to detect changes of GAGs in bone due to concurrent changes of carbonate (CO3) in the similar peak range. This study validates the ability of Raman spectroscopy to detect in situ treatment-, genotype-, and age-related changes in GAG levels of bone matrix.

Corrigendum: PHF20 promotes glioblastoma cell malignancies through a WISP1/BGN-dependent pathway.

[This corrects the article DOI: 10.3389/fonc.2020.573318.].

Expression and prognostic analysis of BGN in head and neck squamous cell carcinoma.

OBJECTIVE: BGN belongs to class of small leucine rich proteoglycans, which is high expression in plenty of human cancers. However, the detailed role of BGN remains unclear in Head and neck squamous cell carcinoma (HNSC). MATERIALS AND METHODS: In this study, we assessed the transcriptional expression, protein expression, prognosis, co-expressed genes, functional enrichment, and hub genes in HNSC patients based on the data published in the following databases: ONCOMINE, GEPIA, GEO, LinkedOmics, and HPA databases. Data from the TCGA database was used to analyze the correlations between BGN expression and different clinicopathological features, as well as prognostic analysis. RESULTS: We found that the expression of BGN is higher in patients with HNSC than in control tissues. Pathologically, high BGN expression was significantly correlated with T3 and T4 stage. Besides, high expression of BGN is a poor prognostic factor for overall surviva, not disease free survival. The co-expression genes associated with BGN expression exhibited enriched in various function and pathway, such as extracellular matrix, mitochondrion, PI3K-Akt signaling pathway. A total of 10 hub genes were identified from the co-expressed genes, within which five genes, including FSTL1, LAMB1, SDC2, VCAN, and IGFBP7, were significantly increased in patient's with HNSC. BGN exhibited weak correlations with tumor-infiltrating CD4+ T, macrophages cell, and dendritic cells. Futhermore, many markers of infiltrating immune cells, such as Treg, showed different BGN-related immune infiltration patterns. BGN expression showed strong correlations with diverse immune marker sets in COAD and STAD. CONCLUSIONS: Our results demonstrated that BGN is high expression in HNSC and is a poor prognostic factor for clinical outcome in patients with HNSC. It could serve as a potential prognostic biomarker for patients survival in HNSC.

Identification of BGN and THBS2 as metastasis-specific biomarkers and poor survival key regulators in human colon cancer by integrated analysis.

BACKGROUND: Colon cancer is the second leading cause of death worldwide. Exploring key regulators in colon cancer metastatic progression could lead to better outcomes for patients. METHODS: Initially, the transcriptional profiles of 681 colonrectal cancer (CRC) cases were used to discover signature genes that were significantly correlated with colon cancer metastasis. These signature genes were then validated using another independent 210 CRC cases' transcriptomics and proteomics profiles, and Kaplan-Meier regression analyses were used to screen the key regulators with patients' survival. Immunohistochemical staining was used to confirm the biomarkers, and transit knockdown was used to explore their implications on colon cancer cells migration and invasion abilities. The impact on the key signalling molecules in epithelial-mesenchymal transition (EMT) process that drive tumour metastasis was tested using Western blot. The response to clinical standard therapeutic drugs was compared to clinical prognosis of key regulators using an ROC plotter. RESULTS: Five genes (BGN, THBS2, SPARC, CDH11 and SPP1) were initially identified as potential biomarkers and therapeutic targets of colon cancer metastasis. The most significant signatures associated with colon cancer metastasis were determined to be BGN and THBS2. Furthermore, highly expression of BGN and THBS2 in tumours was linked to a worse survival rate. BGN and THBS2 knockdown significantly reduced colon cancer cells migration and invasion, as well as down-regulating three EMT-related proteins (Snail, Vimentin and N-cadherin), and increasing the proliferation inhibitory effect of 5-fluorouracil, irinotecan and oxaliplatin treatment. CONCLUSIONS: CRC metastatic progression, EMT phenotypic transition and poor survival time have been linked to BGN and THBS2. They could be utilized as potential diagnostic and therapeutic targets for colon cancer metastatic patients with a better prognosis.

Investigating the mechanophysical and biological characteristics of therapeutic dental cement incorporating copper doped bioglass nanoparticles.

OBJECTIVE: This study was investigated the mechanophysical properties of zinc phosphate cement (ZPC) with or without the copper doped bioglass nanoparticles (Cu-BGn) and their biological effect on dental pulp human cells and bacteria. MATERIALS AND METHODS: Cu-BGn were synthesized and characterized firstly and then, the experimental (Cu-ZPC) and control (ZPC) samples were fabricated with similar sizes and/or dimensions (diameter: 4 mm and height: 6 mm) based on the International Organization of Standards (ISO). Specifically, various concentrations of Cu-BGn were tested, and Cu-BGn concentration was optimized at 2.5 wt% based on the film thickness and overall setting time. Next, we evaluated the mechanophysical properties such as compressive strength, elastic modulus, hardness, and surface roughness. Furthermore, the biological behaviors including cell viability and odontoblastic differentiation by using dental pulp human cells as well as antibacterial properties were investigated on the Cu-ZPC. All data were analyzed statistically using SPSS® Statistics 20 (IBM®, USA). p < 0.05 (*) was considered significant, and 'NS' represents nonsignificant. RESULTS: Cu-BGn was obtained via a sol-gel method and added onto the ZPC for fabricating a Cu-ZPC composite and for comparison, the Cu-free-ZPC was used as a control. The film thickness (≤ 25 µm) and overall setting time (2.5-8 min) were investigated and the mechanophysical properties showed no significance ('NS') between Cu-ZPC and bare ZPC. However, cell viability and odontoblastic differentiation, alkaline phosphate (ALP) activity and alizarin red S (ARS) staining were highly stimulated in the extracts from the Cu-ZPC group compared to the ZPC group. Additionally, the antibacterial test showed that the Cu-ZPC extracts were more effective than the ZPC extracts (p < 0.05). SIGNIFICANCE: Cu-ZPC showed adequate mechanophysical properties (compressive strength, hardness, and surface roughness) and enhanced odontoblastic differentiation as well as antibacterial properties compared to the ZPC-only group. Based on the findings, the fabricated Cu-ZPC might have the potential for use in the field of dental medicine and clinical applications.

Novel regulatory roles of small leucine-rich proteoglycans in remodeling of the uterine cervix in pregnancy.

The cervix undergoes rapid and dramatic shifts in collagen and elastic fiber structure to achieve its disparate physiological roles of competence during pregnancy and compliance during birth. An understanding of the structure-function relationships of collagen and elastic fibers to maintain extracellular matrix (ECM) homeostasis requires an understanding of the mechanisms executed by non-structural ECM molecules. Small-leucine rich proteoglycans (SLRPs) play key functions in biology by affecting collagen fibrillogenesis and regulating enzyme and growth factor bioactivities. In the current study, we evaluated collagen and elastic fiber structure-function relationships in mouse cervices using mice with genetic ablation of decorin and/or biglycan genes as representative of Class I SLRPs, and lumican gene representative of Class II SLRP. We identified structural defects in collagen fibril and elastic fiber organization in nonpregnant mice lacking decorin, or biglycan or lumican with variable resolution of defects noted during pregnancy. The severity of collagen and elastic fiber defects was greater in nonpregnant mice lacking both decorin and biglycan and defects were maintained throughout pregnancy. Loss of biglycan alone reduced tissue extensibility in nonpregnant mice while loss of both decorin and biglycan manifested in decreased rupture stretch in late pregnancy. Collagen cross-link density was similar in the Class I SLRP null mice as compared to wild-type nonpregnant and pregnant controls. A broader range in collagen fibril diameter along with an increase in mean fibril spacing was observed in the mutant mice compared to wild-type controls. Collectively, these findings uncover functional redundancy and hierarchical roles of Class I and Class II SLRPs as key regulators of cervical ECM remodeling in pregnancy. These results expand our understating of the critical role SLRPs play to maintain ECM homeostasis in the cervix.

Probiotics partially attenuate the severity of acute kidney injury through an immunomodulatory effect.

BACKGROUND: A healthy microbiome helps maintain the gut barrier and mucosal immune tolerance. Previously, we demonstrated that acute kidney injury (AKI) provoked dysbiosis, gut inflammation, and increased permeability. Here, we investigated the renoprotective effects of the probiotic Bifidobacterium bifidum BGN4 and the underlying mechanisms thereof. METHODS: C57BL/6 mice were subjected to bilateral renal ischemia-reperfusion injury (IRI) or sham operation. In the probiotic-treated group, BGN4 was administered by gavage once daily, starting 2 weeks before injury. RESULTS: Administration of BGN4 significantly increased gut microbiome diversity and prevented expansion of the Enterobacteriaceae and Bacteroidetes that were the hallmarks of AKI-induced dysbiosis. Further, BGN4 administration also significantly reduced other IRI-induced changes in the colon microenvironment, including effects on permeability, apoptosis of colon epithelial cells, and neutrophil and proinflammatory macrophage infiltration. Mononuclear cells co-cultured with BGN4 expressed significantly increased proportions of CD103+/CD11c+ and CD4+ CD25+ Treg cells, suggesting a direct immunomodulatory effect. BGN4 induced Treg expansion in colon, mesenteric lymph nodes (MNL), and kidney. BGN4 also reduced CX3CR1intermediateLy6Chigh monocyte infiltration and interleukin (IL)-17A suppression in the small intestine, which may have attenuated AKI severity, kidney IL-6 messenger RNA expression, and AKI-induced liver injury. CONCLUSION: Prior supplementation with BGN4 significantly attenuated the severity of IRI and secondary liver injury. This renoprotective effect was associated with increased Foxp3 and reduced IL-17A expression in the colon, MNL, and kidney, suggesting that BGN4-induced immunomodulation might contribute to its renoprotective effects. Probiotics may therefore be a promising strategy to reduce AKI severity and/or remote organ injury.

Prognostic and Predictive Value of BGN in Colon Cancer Outcomes and Response to Immunotherapy.

BACKGROUND: Colon cancer is one of the most frequent malignancies and causes high mortality worldwide. Exploring the tumor-immune interactions in the tumor microenvironment and identifying new prognostic and therapeutic biomarkers will assist in decoding the novel mechanism of tumor immunotherapy. BGN is a typical extracellular matrix protein that was previously validated as a signaling molecule regulating multiple processes of tumorigenesis. However, its role in tumor immunity requires further investigation. METHODS: The differentially expressed genes in three GEO datasets were analyzed, and BGN was identified as the target gene by intersection analysis of PPIs. The relevance between clinical outcomes and BGN expression levels was evaluated using data from the GEO database, TCGA and tissue microarray of colon cancer samples. Univariable and multivariable Cox regression models were conducted for identifying the risk factors correlated with clinical prognosis of colon cancer patients. Next, the association between BGN expression levels and the infiltration of immune cells as well as the process of the immune response was analyzed. Finally, we predicted the immunotherapeutic response rates in the subgroups of low and high BGN expression by TIS score, ImmuCellAI and TIDE algorithms. RESULTS: BGN expression demonstrated a statistically significant upregulation in colon cancer tissues than in normal tissues. Elevated BGN was associated with shorter overall survival as well as unfavorable clinicopathological features, including tumor size, serosa invasion and length of hospitalization. Mechanistically, pathway enrichment and functional analysis demonstrated that BGN was positively correlated with immune and stromal scores in the TME and primarily involved in the regulation of immune response. Further investigation revealed that BGN was strongly expressed in the immunosuppressive phenotype and tightly associated with the infiltration of multiple immune cells in colon cancer, especially M2 macrophages and induced Tregs. Finally, we demonstrated that high BGN expression presented a better immunotherapeutic response in colon cancer patients. CONCLUSION: BGN is an encouraging predictor of diagnosis, prognosis and immunotherapeutic response in patients with colon cancer. Assessment of BGN expression represents a novel approach with great promise for identifying patients who may potentially benefit from immunotherapy.

Lysed and disrupted Bifidobacterium bifidum BGN4 cells promote anti-inflammatory activities in lipopolysaccharide-stimulated RAW 264.7 cells.

Bifidobacterium bifidum BGN4 has been shown to improve the immune system by regulating interleukin (IL)-6 in RAW 264.7 macrophage cells. In this study, the dead cells of B. bifidum BGN4 were produced by enzymatic and physical processing to enhance the inhibition properties of pro-inflammatory cytokines using lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Notably, the secretion levels of cytokines such as interleukin (IL)-1ß, IL-6, IL-8, and tumor necrosis factor (TNF)-α were decreased by the cell-wall disrupted extracts compared to heat-killed cells. The result suggests that the exposed interior-surface of B. bifidum BGN4 has a potential ability to regulate the immune-responses in the gastrointestinal tract due to major substances in inside-cell wall such as peptidoglycan and teichoic acids. In conclusion, the lysed and disrupted cells from the inside out of B. bifidum BGN4 have anti-inflammatory properties as paraprobiotic agents to control chronic inflammatory related-diseases.

LINC00460 Promotes Cell Proliferation, Migration, Invasion, and Epithelial-Mesenchymal Transition of Head and Neck Squamous Cell Carcinoma via miR-320a/BGN Axis.

PURPOSE: Long non-coding RNAs (lncRNAs) play critical roles in cancer onset and development, including head and neck squamous cell carcinoma (HNSCC). This study aimed to investigate the biological role of LINC00460 and the mechanisms underlying epithelial-mesenchymal transition (EMT) in HNSCC. METHODS: Aberrantly LINC00460 expression in HNSCC and overall survival outcomes were constructed using the TCGA database. Quantitative real-time polymerase chain reaction (RT-qPCR) was applied to examine the LINC00460 expression level in HNSCC cell lines. The role of LINC00460 knockdown on HNSCC cell growth, migration, invasion, and EMT was investigated in vitro using cell counting kit-8 (CCK-8), colony formation, transwell assay, and Western blot assay. Besides, bioinformatics prediction, dual-luciferase reporter assay, and RNA immunoprecipitation (RIP) were performed to reveal the interaction among LINC00460 and its target genes. The function of the LINC00460/miR-320a/BGN axis in HNSCC cells was clarified by rescue assays. Furthermore, the in vivo effects of LINC00460 on tumor growth were investigated using mice xenograft models. RESULTS: In this study, LINC00460 was upregulated in HNSCC tissues and cells and was associated with poor clinical prognosis. Further functional analysis showed that LINC00460 knockdown decreased HNSCC cell proliferation, migration, invasion, as well as EMT in vitro. Mechanistic investigation indicated that LINC00460 sponged miR-320a to upregulate Biglycan (BGN) expression, thereby facilitating HNSCC progression and induced EMT. Moreover, knockdown of LINC00460 significantly suppressed the progression of HNSCC cells in vivo. CONCLUSION: Taken together, LINC00460 mediates miR-320a/BGN signaling axis to promote cell proliferation, migration, invasion, and induce the EMT process in HNSCC cells. Our findings elucidated a novel mechanism underlying the progression of HNSCC. LINC00460 could serve as a potential therapeutic target for the treatment of HNSCC.

Identification and Analysis of Key Genes Driving Gastric Cancer Through Bioinformatics.

Objective: The aim of this study was to use bioinformatic analyses to identify key genes and pathways driving gastric cancer (GC). Materials and Methods: The gene expression profiles, from human gastric tissue samples were downloaded from the Gene Expression Omnibus (GSE)29272 dataset. These data revealed 284 differentially expressed genes (DEGs) that included a group upregulated in cancer tissues (n = 142) and another group that were downregulated in cancer tissues. (n = 142). These DEGs were identified using the GEO2R. We used multiple online analysis tools, including, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), protein-protein interaction networks, gene expression profiling interactive analysis (GEPIA), and the cBio Cancer Genomics Portal (cBioportal) database. Next, we identified the most significant DEGs using the Kaplan-Meier plotter (KM-plotter) database. Multiple bioinformatic platforms were used to identify candidate prognostic marker genes. We then analyzed freshly frozen GC tissues for the expression of these marker genes to validate the informatic findings. Results: We identified three DEGs related to overall survival from our analyses of the GEO data. Next, we analyzed these three DEGs in GEPIA and the cBioportal database and found that the biglycan (BGN) gene was related to invasion and metastases of GCs. This finding of differential gene expression was confirmed in a separate laboratory analysis of normal and GC tissues. In this analysis we found that high levels of BGN expression were correlated with GC clinicopathological characteristics, including microvascular tumor thrombus (p = 0.018), lymph node metastases (p = 0.013), and vessel invasion (p = 0.004). Conclusions: BGN expression levels appear to be an independent prognostic factor for predicting the survival times of GC patients.

BGN and COL11A1 Regulatory Network Analysis in Colorectal Cancer (CRC) Reveals That BGN Influences CRC Cell Biological Functions and Interacts with miR-6828-5p.

PURPOSE: We explored specific expression profiles of BGN and COL11A1 genes and studied their biological functions in CRC using bioinformatics tools. PATIENTS AND METHODS: A total of 68 pairs of cancer and non-cancerous tissues from CRC patients were enrolled in this study. Methods we used in this articles including: qRT-PCR, Western blot analysis, ELISA, GO and KEGG regulatory network analysis, tumor infiltration, luciferase reporter-based protein and etc. RESULTS: According to The Cancer Genome Atlas (TCGA) data, BGN and COL11A1 expression levels were significantly higher in CRC patient samples than in samples from healthy controls. Moreover, levels were much higher in late-stage CRC than in early-stage disease, warranting evaluation of these genes as CRC prognostic biomarkers. Subsequently, qRT-PCR, Western blot analysis, and ELISA results obtained from analyses of CRC cells, tissues, and patient sera aligned with TCGA results. GO and KEGG regulatory network analysis revealed BGN- and COL11A1-associated genes that were functionally related to extracellular matrix (ECM) receptor pathway activation, with transcription factor genes RELA and NFKB1 positively associated with BGN expression and CEBPZ and SIRT1 with COL11A1 expression. Meanwhile, BGN and COL11A1 expression were separately and significantly correlated to tumor infiltration by six immune cell types. Additionally, kinase genes PLK1 and LYN appeared to be downstream targets of differentially expressed BGN and COL11A1, respectively. In addition, the expression of PLK1 mRNA was down-regulated while BGN was down-regulated. Finally, BGN effects on CRC cell proliferation, cycle, apoptosis, invasion, and migration were studied using molecular biological methods, including luciferase reporter-based protein analysis, qRT-PCR, and Western blot results, which revealed that miR-6828-5p may regulate BGN expression. CONCLUSION: We speculate that the use of BGN and COL11A1 as CRC biomarkers would improve CRC staging, while also providing several novel targets for use in the development of more effective CRC treatments.

PHF20 Promotes Glioblastoma Cell Malignancies Through a WISP1/BGN-Dependent Pathway.

Glioblastoma (GBM) stem cells are resistant to cancer therapy, and therefore responsible for tumor progression and recurrence after conventional therapy. However, the molecular mechanisms driving the maintenance of stemness and dedifferentiation are poorly understood. In this study, we identified plant homeodomain finger-containing protein 20 (PHF20) as a crucial epigenetic regulator for sustaining the stem cell-like phenotype of GBM. It is highly expressed in GBM and tightly associated with high levels of aggressiveness of tumors and potential poor prognosis in GBM patients. Knockout of PHF20 inhibits GBM cell proliferation, as well as its invasiveness and stem cell-like traits. Mechanistically, PHF20 interacts with WDR5 and binds to the promoter regions of WISP1 for its expression. Subsequently, WISP1 and BGN act in concert to regulate the degradation of ß-Catenin. Our findings have identified PHF20 as a key driver of GBM malignant behaviors, and provided a potential target for developing prognosis and therapy.

Identification of hub genes in colon cancer via bioinformatics analysis.

OBJECTIVES: This study aimed to investigate hub genes and their prognostic value in colon cancer via bioinformatics analysis. METHODS: Differentially expressed genes (DEGs) of expression profiles (GSE33113, GSE20916, and GSE37364) obtained from Gene Expression Omnibus (GEO) were identified using the GEO2R tool and Venn diagram software. Function and pathway enrichment analyses were performed, and a protein-protein interaction (PPI) network was constructed. Hub genes were verified based on The Cancer Genome Atlas (TCGA) and Human Protein Atlas (HPA) databases. RESULTS: We identified 207 DEGs, 62 upregulated and 145 downregulated genes, enriched in Gene Ontology terms "organic anion transport," "extracellular matrix," and "receptor ligand activity", and in the Kyoto Encyclopedia of Genes and Genomes pathway "cytokine-cytokine receptor interaction." The PPI network was constructed and nine hub genes were selected by survival analysis and expression validation. We verified these genes in the TCGA database and selected three potential predictors (ZG16, TIMP1, and BGN) that met the independent predictive criteria. TIMP1 and BGN were upregulated in patients with a high cancer risk, whereas ZG16 was downregulated. The immunostaining results from HPA supported these findings. CONCLUSION: This study indicates that these hub genes may be promising prognostic indicators or therapeutic targets for colon cancer.

BGN/TLR4/NF-B Mediates Epigenetic Silencing of Immunosuppressive Siglec Ligands in Colon Cancer Cells.

Human Toll-like receptor (TLR) signaling plays a vital role in intestinal inflammation by activating the NF-B pathway. By querying GENT2 datasets, we identified the gene expression level of TLR2 and TLR4 as being substantially increased in colorectal cancer. Introduction of shRNAs for TLR4 but not TLR2 dramatically recovered disialyl Lewisa and sialyl 6-sulfo Lewisx glycans, which are preferentially expressed in non-malignant colonic epithelial cells and could serve as ligands for the immunosuppressive molecule Siglec-7. We screened several TLR4 ligands and found that among them BGN is highly expressed in cancers and is involved in the epigenetic silencing of Siglec-7 ligands. Suppression of BGN expression substantially downregulated NF-B activity and the marker H3K27me3 in the promoter regions of the SLC26A2 and ST6GalNAc6 genes, which are involved in the synthesis of those glycans, and restored expression of normal glycans as well as Siglec-7 binding activities. We show that in the presence of TLR4, inflammatory stimuli initiate a positive loop involving NF-B that activates BGN and further enhances TLR4 activity. Present findings indicate a putative mechanism for the promotion of carcinogenesis by loss of immunosuppressive ligands by the BGN/TLR4/ NF-B pathway.