Comprehensive analysis of expression and prognostic value of the claudin family in human breast cancer.

Claudins (CLDN) are structural components of tight junctions that function in paracellular transport and maintain the epithelial barrier function. Altered expression and distribution of members of the claudin family have been implicated in several cancers including breast cancer (BC). We performed a comprehensive analysis of the expression and prognostic value of claudins in BC using various online databases. Compared with normal tissues, CLDN3, 4, 6, 7, 9, and 14 were upregulated in BC tissues, whereas CLDN2, 5, 8, 10, 11, 15, 19, and 20 were downregulated. A high expression of CLDN2, 5, 6, 9, 10, 11, and 14-20 was associated with better relapse-free survival (RFS), whereas a high CLDN3 expression correlated with poor RFS. In addition, a high expression of CLDN3, 4, 14, and 20 was associated with poor overall survival (OS), whereas that of CLDN5 and CLDN11 was linked to a better OS. Although METABRIC and TCGA datasets revealed 22% and 27% gene alterations, respectively, in the members of the claudin family, these were not associated with survival. These findings suggest CLDN3, 5, and 11 could be promising therapeutic targets for patients with BC.

Identification of Key Genes Involved in Diabetic Peripheral Neuropathy Progression and Associated with Pancreatic Cancer.

INTRODUCTION: Diabetes mellitus (DM) patients suffer from high morbidity and premature mortality due to various diabetic complications and even cancers. Therefore, this study aimed to identify key genes involved in the pathogenesis of diabetic peripheral neuropathy (DPN) and pancreatic cancer (PC). METHODS: We analyzed three gene expression profiles (GSE95849, GSE28735 and GSE59953) to obtain differentially expressed genes (DEGs). Then, Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed by using the Database for Annotation, Visualization, and Integrated Discovery (DAVID). The Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database was then used to establish a protein-protein interaction (PPI) network. The MCODE and cytoHubba plug-ins of Cytoscape were used to select hub genes. Finally, survival analysis of the hub genes was performed using the Kaplan-Meier plotter and GEPIA online tool. RESULTS: We first analyzed GSE95849 to obtain DPN-related genes. DEGs were obtained from three groups in GSE95849. The DEGs were enriched in the Toll-like receptor signaling pathway, hematopoietic cell lineage and chemokine signaling pathway. Importantly, we identified three shared genes as hub genes, including TLR4, CCR2 and MMP9. We then analyzed and integrated GSE95849 and GSE28735 to obtain genes common in DM and PC. A total of 58 mutual DEGs were identified, and these DEGs were enriched in the ECM-receptor interaction, focal adhesion and pathways in cancer. Five hub genes (including PLAU, MET, CLU, APOL1 and MMP9) were associated with the overall survival of PC patients. However, the results from the analysis of GSE59953 showed that hyperglycemia or TGF-ß1 treatment did not affect the expression level of these hub genes, but the DEGs based on hyperglycemia or TGF-ß1 treatment were mostly enriched in the ECM-receptor interaction, focal adhesion and pathways in cancer. Finally, functional enrichment analysis of MMP9 showed that significant genes correlated with MMP9 were associated with the tumorigenicity of cancers, insulin resistance, development of DM and inflammation. CONCLUSION: In summary, inflammation and immunity-related pathways may play an important role in DM and DPN, while the ECM-receptor interaction, focal adhesion and pathways in cancer pathways may play significant roles in DM and PC. MMP9 may be used as a prognostic marker for PC and may be helpful for the treatment of DM, DPN and PC.

Corrigendum to "Bioinformatics Analysis of Potential Key Genes in Trastuzumab-Resistant Gastric Cancer".

[This corrects the article DOI: 10.1155/2019/1372571.].

Bioinformatics Analysis of Potential Key Genes in Trastuzumab-Resistant Gastric Cancer.

BACKGROUND: This study was performed to identify genes related to acquired trastuzumab resistance in gastric cancer (GC) and to analyze their prognostic value. METHODS: The gene expression profile GSE77346 was downloaded from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) were obtained by using GEO2R. Functional and pathway enrichment was analyzed by using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Search Tool for the Retrieval of Interacting Genes (STRING), Cytoscape, and MCODE were then used to construct the protein-protein interaction (PPI) network and identify hub genes. Finally, the relationship between hub genes and overall survival (OS) was analyzed by using the online Kaplan-Meier plotter tool. RESULTS: A total of 327 DEGs were screened and were mainly enriched in terms related to pathways in cancer, signaling pathways regulating stem cell pluripotency, HTLV-I infection, and ECM-receptor interactions. A PPI network was constructed, and 18 hub genes (including one upregulated gene and seventeen downregulated genes) were identified based on the degrees and MCODE scores of the PPI network. Finally, the expression of four hub genes (ERBB2, VIM, EGR1, and PSMB8) was found to be related to the prognosis of HER2-positive (HER2+) gastric cancer. However, the prognostic value of the other hub genes was controversial; interestingly, most of these genes were interferon- (IFN-) stimulated genes (ISGs). CONCLUSIONS: Overall, we propose that the four hub genes may be potential targets in trastuzumab-resistant gastric cancer and that ISGs may play a key role in promoting trastuzumab resistance in GC.

4-Hydroxyisoleucine ameliorates an insulin resistant-like state in 3T3-L1 adipocytes by regulating TACE/TIMP3 expression.

BACKGROUND: Obesity-associated insulin resistance (IR) is highly correlated with soluble tumor necrosis factor-α (sTNF-α), which is released from transmembranous TNF-α by TNF-α converting enzyme (TACE). In vivo, TACE activity is suppressed by tissue inhibitor of metalloproteinase 3 (TIMP3). Agents that can interact with TACE/TIMP3 to improve obesity-related IR would be highly valuable. In the current study, we assessed whether (2S,3R,4S)-4-hydroxyisoleucine (4-HIL) could modulate TACE/TIMP3 and ameliorate an obesity-induced IR-like state in 3T3-L1 adipocytes. MATERIALS AND METHODS: 3T3-L1 adipocytes were incubated in the presence of 25 mM glucose and 0.6 nM insulin to induce an IR-like state, and were then treated with different concentrations of 4-HIL or 10 µM pioglitazone (positive control). The glucose uptake rate was determined using the 2-deoxy-[(3)H]-D-glucose method, and the levels of sTNF-α in the cell supernatant were determined using ELISA. The protein expression of TACE, TIMP3, and insulin signaling-related molecules was measured using western blotting. RESULTS: Exposure to high glucose and insulin for 18 hours increased the levels of sTNF-α in the cell supernatant. The phosphorylation of insulin receptor substrate-1 (IRS-1) Ser(307) and Akt Ser(473) was increased, whereas the protein expression of IRS-1, Akt, and glucose transporter-4 was decreased. The insulin-induced glucose uptake was reduced by 67% in 3T3-L1 adipocytes, which indicated the presence of an IR-like state. The above indexes, which demonstrated the successful induction of an IR-like state, were reversed by 4-HIL in a dose-dependent manner by downregulating and upregulating the protein expression of TACE and TIMP3 proteins, respectively. CONCLUSION: 4-HIL improved an obesity-associated IR-like state in 3T3-L1 adipocytes by targeting TACE/TIMP3 and the insulin signaling pathway.

4-Hydroxyisoleucine improves insulin resistance in HepG2 cells by decreasing TNF-α and regulating the expression of insulin signal transduction proteins.

Previous studies have indicated that 4­hydroxyisoleucine (4­HIL) improves insulin resistance, however, the underlying mechanisms remain to be elucidated. In the present study, the molecular mechanisms underlying how 4­HIL improves insulin resistance in hepatocytes were examined. HepG2 cells were co­cultured with insulin and a high glucose concentration to obtain insulin­resistant (IR) HepG2 cells. Insulin sensitivity was determined by measuring the glucose uptake rate. The IR HepG2 cells were treated with different concentrations of 4­HIL to determine its effect on IR Hep2 cells. The levels of tumor necrosis factor­α (TNF­α) were measured by an enzyme­linked immunosorbent assay and protein levels of TNF­α converting enzyme (TACE)/tissue inhibitor of metalloproteinase 3 (TIMP3), insulin receptor substrate (IRS)­1, IRS­2, phosphorylated (p)­IRS­1 (Ser307) and glucose transporter type 4 (GLUT4) were measured by western blot analysis. The results of the present study demonstrated that insulin­induced glucose uptake was reduced in IR HepG2 cells; however, this reduction was reversed by 4­HIL in a dose­dependent manner. 4­HIL achieved this effect by downregulating the expression of TNF­α and TACE, and upregulating the expression of TIMP3 in IR HepG2 cells. In addition, 4­HIL increased the expression of the insulin transduction regulators IRS­1 and GLUT4, and decreased the expression of p­IRS­1 (Ser307), without affecting the expression of IRS­2. The present study suggests that 4­HIL improved insulin resistance in HepG2 cells by the following mechanisms: 4­HIL reduced TNF­α levels by affecting the protein expression of the TACE/TIMP3 system and 4­HIL stimulated the expression of IRS­1 and GLUT4, but inhibited the expression of p­IRS­1 (Ser307).

4-Hydroxyisoleucine improves hepatic insulin resistance by restoring glycogen synthesis in vitro.

INTRODUCTION/AIMS: Hydroxyisoleucine (4-HIL), derived from fenugreek seeds, improves insulin resistance in 3T3-L1 adipocytes and L6 myotubes. However, the effects of 4-HIL on liver glycogen synthesis and hepatic insulin resistance have not been described. The aim of this study was to investigate the effects of 4-HIL on glycogen synthesis in a tumor necrosis factor-α (TNF-α)-induced insulin resistance model using HepG2 cells. MATERIALS AND METHODS: HepG2 cells were divided into eight groups: control; TNF-α; and 5, 10, or 20 µM 4-HIL without or with TNF-α. Glycogen and protein expression were evaluated using a glycogen assay kit and western blotting, respectively. RESULTS: Glycogen levels did not differ between the 4-HIL groups and control (P>0.05), but were decreased significantly in the TNF-α group (P<0.05), indicating the establishment of insulin-resistant HepG2 cells. Adding 20 µM 4-HIL to TNF-α-treated cells increased glycogen levels (P<0.05). Relative to the control group, the P-IRS-1/IRS-1 and P-JNK/JNK ratios were increased (P<0.001) in the TNF-α group, whereas the P-AKT/AKT and P-GSK/GSK ratios were decreased (P<0.001). When 20 µM 4-HIL was added to TNF-α-treated cells, the P-IRS-1/IRS-1 and P-JNK/JNK ratios decreased (P<0.001 and P<0.05, respectively), whereas the P-AKT/AKT and P-GSK/GSK ratios increased (P<0.05 and P<0.001). CONCLUSIONS: 4-HIL directly or indirectly reversed TNF-α reduced glycogen levels by inhibiting JNK and IRS-1 (Ser(307)) phosphorylation and increasing AKT (Ser(473)) and GSK-3 phosphorylation. These findings demonstrate that 4-HIL modulates hepatic insulin resistance at the molecular level, and suggest that it is a novel potential therapeutic agent for the treatment of insulin resistance in patients with diabetes.

[Effect of Trigonella foenum-graecum 4-hydroxyisoleucine on high-glucose induced insulin resistance in 3T3-L1 adipocytes of mice].

OBJECTIVE: To investigate the effect of 4-hydroxyisoleucine (4-HIL), an active component of Trigonella Foenum-graecum L. on high glucose induced insulin resistance (IR) in 3T3-L1 adipocytes, and to explore underlying molecular mechanisms. METHODS: 3T3-L1 adipocytes were treated with 25 mmol/L glucose and 0.6 nmol/L insulin to induce IR. They were intervened by different concentrations of 4-HIL (at 5, 10, and 20 micromol/L). [3H]-Deoxy-D-glucose up-taking method was used to detect the glucose uptake. The mRNA expression of cellular tumor necrosis factor-alpha (TNF-alpha) was detected by polymerase chain reaction (PCR). The content of TNF-alpha in the culture supernatant was detected by enzyme-linked immunosorbent assay (ELISA). Palmitic acid (PA) acted as the control. RESULTS: After intervened by 25 mmol/L glucose and 0.6 nmol/L insulin for 18 h, the insulin-stimulated glucose transportation in 3T3-L1 adipocytes was inhibited by 63%. The mRNA expression of cellular TNF-alpha in adipocytes significantly increased, when compared with that in normal adipocytes (P < 0.05). The level of TNF-alpha secreted in the culture supernatant was increased by 70 pg/mL (P < 0.05). Similar changes occurred in the PA group. After exposure to 4-HIL (5, 10, or 20 micromol/L) for 24 h, the glucose transportation was increased by 35%, 50%, and 60%, respectively. PCR results showed that along with increasing 4-HIL concentrations, the mRNA expression of cellular TNF-alpha showed a decreasing trend, showing statistical difference when compared with the model group and the PA group (P < 0.05). Compared with the model group, the TNF-alpha level in the supernatant was respectively reduced by 10 pg/mL, 18 pg/mL, and 39 pg/mL after intervention (P < 0.05). CONCLUSION: 4-HIL could remarkably improve high glucose-induced IR in 3T3-L1 adipocytes. Meanwhile, 4-HIL could inhibit the secretion of TNF-alpha.