CYP4F12 is a potential biomarker and inhibits cell migration of head and neck squamous cell carcinoma via EMT pathway.

Head and neck squamous cell carcinoma (HNSC) is the most common malignant tumor of head and neck. Due to the insidious nature of HNSC and the lack of effective early diagnostic indicators, the development of novel biomarkers to improve patient prognosis is particularly urgent. In this study, we explored and validated the correlation between cytochrome P450 family 4 subfamily F member 12 (CYP4F12) expression levels and HNSC progression using data from The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO) datasets and collected patient samples. We analyzed the association of CYP4F12 expression with clinicopathological features, immune correlation and prognosis. Finally, we analyzed the correlation between CYP4F12 and pathways, and verified by experiments. The results showed that CYP4F12 was low expressed in tumor tissues, participated in a variety of phenotypic changes of HNSC and affected immune cell infiltration. Pathway analysis indicated that CYP4F12 may play a key role in tumor cell migration and apoptosis. Experimental results showed that over-expression of CYP4F12 inhibited cell migration and enhanced the adhesion between cells and matrix by inhibiting epithelial-mesenchymal transition (EMT) pathway in HNSC cells. In conclusion, our study provided insights into the role of CYP4F12 in HNSC and revealed that CYP4F12 may be a potential therapeutic target for HNSC.

Comprehensive analysis reveals CCDC60 as a potential biomarker correlated with prognosis and immune infiltration of head and neck squamous cell carcinoma.

Background: Coiled-coil domain containing 60 (CCDC60) is a member of the CCDC family, which participates in the progression of many types of cancer. However, the prognostic value of CCDC60 in head and neck squamous cell carcinoma (HNSC) and its function in tumor immunity remain unclear. Methods: CCDC60 expression and its prognostic potential in HNSC were evaluated by bioinformatics approaches, which was validated in human HNSC samples. Genetic alteration analysis of CCDC60 and the underlying biological function of CCDC60 related co-expressed genes in HNSC were analyzed. The impact of CCDC60 on the regulation of immune infiltration in HNSC was comprehensively investigated. In vitro, a series of functional assays on CCDC60 were performed in HNSC cells. Results: Our study has indicated that compared with the adjacent normal tissues, CCDC60 expression was considerably downregulated in HNSC tissues. High CCDC60 expression was connected with favorable outcome of HNSC patients, and its prognostic significance was examined by distinct clinical characteristics. We identified the CCDC60-related co-expression genes, which were mainly enriched in the NOD-like receptor signaling pathway associated with the inhibition of tumor growth, leading to a better prognosis of HNSC patients. In vitro, CCDC60 overexpression significantly inhibited the growth, migration and invasiveness but regulated cell cycle progression, and promoted cell adhesion of Fadu and Cal27 cells. Additionally, high CCDC60 expression had strong connections with the infiltrating levels of immune cells, immune marker sets, immunomodulators and chemokines in HNSC, suggesting that targeting CCDC60 could be a promising strategy to enhance the efficacy of immunotherapy for HNSC patients. Conclusion: Tumor suppressor CCDC60 may be identified as a prognostic and immune-related indicator in HNSC, which had the potential functions in regulating the immune infiltration of HNSC and improving the response to immunotherapy for HNSC patients.

Identify metabolism-related genes IDO1, ALDH2, NCOA2, SLC7A5, SLC3A2, LDHB, and HPRT1 as potential prognostic markers and correlate with immune infiltrates in head and neck squamous cell carcinoma.

Hypopharyngeal squamous cell carcinoma (HSCC) is a kind of head and neck squamous cell carcinoma (HNSCC) with poor prognosis. Metabolic reprogramming may regulate the tumor microenvironment (TME) by adapting quickly to cellular stress and regulating immune response, but its role in HSCC has not been reported. We used the nCounter® Metabolic Pathways Panel to investigate metabolic reprogramming, cellular stress, and their relationship in HSCC tissues and adjacent normal tissues. Metabolism-related pathways nucleotide synthesis and glycolysis pathways were significantly upregulated, while amino acid synthesis and fatty acid oxidation pathways were significantly downregulated in HSCC tissues compared to adjacent normal tissues. There is a significant correlation between metabolism-related pathways and cellular stress pathways. Enrichment of immune cell and tumor infiltrating lymphocyte (TIL) analysis showed changes in immune responses between HSCC tissues and adjacent normal tissues. Overall survival analysis showed that upregulated genes CD276, LDHB, SLC3A2, EGFR, SLC7A5, and HPRT1 are potential unfavorable prognostic markers in HNSCC, while downregulated genes EEA1, IDO1, NCOA2, REST, CCL19, and ALDH2 are potential favorable prognostic markers in HNSCC. Moreover, metabolism-related genes IDO1, ALDH2, NCOA2, SLC7A5, SLC3A2, LDHB, and HPRT1 are correlated with immune infiltrates in HNSCC. These results suggest that metabolic reprogramming occurs and correlates with cellular stress and immune response in HSCC, which may help researchers understand mechanisms of metabolic reprogramming and develop effective immunotherapeutic strategies in HNSCC.

Let-7c increases BACE2 expression by RNAa and decreases Aß production.

MicroRNAs (miRNAs) are highly conserved, non-coding transcripts that regulate gene expression in various ways. Evidence suggests that miRNAs may be a contributory factor in neurodegeneration, including Alzheimer's disease (AD), Parkinson's disease (PD), and triplet repeat disorders. In order to further understand the potential roles of miRNAs in the pathogenesis of AD, we analyzed Down syndrome (DS), a special model of AD, by using a TaqMan microRNA array and found that miRNA let-7c was up-regulated in both DS and AD. ELISA assay showed that let-7c reduced the expression level of Aß significantly. Real-time quantitative-polymerase chain reaction (RT-qPCR) was conducted to reveal that the expression level of let-7c increased dramatically in DS cells, patients with DS and mice with AD compared with normal ones respectively. Additionally, western blotting illustrated that let-7c suppressed the expression of Aß by inducing BACE2 to cut C99 and increase the content of C83/80. BACE2 expression was inhibited by let-7c and luciferase reporter gene assay revealed that let-7c increased the activity of wild-type BACE2 promoter but not 3'UTR. Furthermore, promoter analysis of BACE2 confirmed that let-7c could bind to BACE2 in the sequence between -1368 and -1347. In addition, immunoblotting assay demonstrated that let-7c induced BACE2 expression by RNAa. To the best of our knowledge, our study revealed for the first time that let-7c up-regulated BACE2 expression and decreased Aß production.

DYRK1A activates NFATC1 to increase glioblastoma migration.

Glioblastoma (GBM) is the most aggressive glioma, and is prone to develop resistance to chemotherapy and radiotherapy; hence, patients with glioblastoma have a high recurrence rate and a low 1-year survival rate. In addition, the pathogenesis of glioblastoma is complex and largely unknown, and the available treatments are limited. Here, we uncovered a fundamental role of DYRK1A in regulating NFATC1 in GBMs. We found that DYRK1A was highly expressed in glioma and glioblastoma cells, and its expression was positively correlated with that of NFATC1. Moreover, inhibition of DYRK1A promoted NFATC1 degradation in GBM cells and sharply reduced the transactivation of NFATC1, not only by decreasing the expression of NFATC1-targeted genes, but also by reducing the luciferase activity, and vice versa. However, DYRK1A had the opposite effect on NFATC2. Most importantly, our data suggest that DYRK1A inhibition reduces glioblastoma migration. Polypeptides derived from the DYRK1A-targeted motif of NFATC1, by competitively blocking DYRK1A kinase activity on NFATC1, clearly destabilized NFATC1 protein and impaired glioblastoma migration. We propose that the recovery of NFATC1 stability is a key oncogenic event in a large proportion of gliomas, and pharmacological inhibition of DYRK1A by polypeptides could represent a promising therapeutic intervention for GBM.

GPR12 inhibits migration and promotes apoptosis in esophageal cancer and hypopharyngeal cancer cells.

BACKGROUND: G protein-coupled receptor 12 (GPR12) is an orphan receptor with no confirmed endogenous ligands. It plays important roles in both physiological and pathological conditions such as neurogenesis and neural inflammation. However, it remains unclear whether GPR12 regulates carcinogenesis and progression in head and neck squamous cell carcinoma (HNSCC), such as esophageal cancer (EC) and hypopharyngeal cancer (HC). METHODS: The Cancer Genome Atlas (TCGA) database was applied to explore the expression of GPR12. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of GPR12 in cancer tissues. Wound healing and transwell assays were carried out to verify the effect of GPR12 on cell migration. Flow cytometric analysis and caspase-Glo 3/7 assay were carried out to verify the influence of GPR12 on cell apoptosis. Western blotting was used to measure the expression of proteins related to migration and apoptosis. RESULT: The qRT-PCR analyses showed that the expression of GPR12 decreased in EC and HC than that in their paired adjacent normal tissues. Wound healing assay and transwell assay demonstrated that GPR12 inhibited tumor cell migration. Flow cytometry analysis and Caspase-Glo 3/7 Assay suggested that GPR12 promoted apoptosis. The mechanism of GPR12 may function via modulating caspase-7, E-cadherin, and α-catenin in EC and HC cells. CONCLUSION: In conclusion, GPR12 induced apoptosis by activating caspase-7 and inhibited migration through epithelial-to-mesenchymal transition (EMT) in EC and HC. Our findings demonstrated that GPR12 as a potential tumor suppressor mediated cell migration and apoptosis in EC and HC.

Activation of Wnt/ß-catenin signaling restores insulin sensitivity in insulin resistant neurons through transcriptional regulation of IRS-1.

Aberrant expression and phosphorylation of insulin receptor substrate 1 (IRS-1) contribute to brain insulin resistance. However, the underlying mechanism remains elusive. The insulin signaling and Wnt/ß-catenin signaling are two critical pathways for normal cellular function, which interact in both peripheral tissues and the brain and may contribute to insulin resistance. In this study, we aimed to investigate the regulation of IRS-1 and its downstream insulin signaling by Wnt/ß-catenin signaling in primary neurons. We found that the Wnt agonist Wnt3a enhances the insulin signaling in neurons at the basal state via up-regulation of IRS-1. Moreover, Wnt3a up-regulates IRS-1 expression and effectively ameliorates insulin resistance in rat primary neurons induced by chronic high insulin exposure. The insulin-mediated glucose uptake is also stimulated by Wnt3a at both basal and insulin resistant states. We observed that Wnt activation up-regulates IRS-1 gene transcription and the subsequent protein expression in SH-SY5Y cells and rat primary neurons via different means of Wnt/ß-catenin signaling activation, including S33Y ß-catenin over-expression, CHIR99021 and Wnt3a treatment. We further clarified the molecular mechanism of IRS-1 transcriptional activation by Wnt/ß-catenin signaling. The Wnt transcription factor TCF4 binds to the -529 bp to -516 bp of the human IRS-1 promoter fragment and activates IRS-1 transcription. Overall, these data suggested that Wnt/ß-catenin signaling positively regulates IRS-1 and insulin signaling and protects against insulin resistance in neurons.

Dual-specificity tyrosine phosphorylation-regulated kinase 1A ameliorates insulin resistance in neurons by up-regulating IRS-1 expression.

Insulin resistance in the brain is a pathological mechanism that is shared between Alzheimer's disease (AD) and type 2 diabetes mellitus (T2DM). Although aberrant expression and phosphorylation of insulin receptor substrate 1 (IRS-1) contribute to insulin resistance, the underlying mechanism remains elusive. In this study, we used several approaches, including adeno-associated virus-based protein overexpression, immunoblotting, immunoprecipitation, immunohistochemistry, and in situ proximal ligation assays, to investigate the function of dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) in IRS-1 regulation and the downstream insulin signaling in neurons. We found that DYRK1A overexpression up-regulated IRS-1 expression by slowing turnover of the IRS-1 protein. We further observed that DYRK1A directly interacted with IRS-1 and phosphorylated IRS-1's multiple serine residues. Of note, DYRK1A and IRS-1 were coordinately up-regulated in the prefrontal cortex of db/db mice brain. Furthermore, DYRK1A overexpression ameliorated chronic high insulin-induced insulin resistance in SH-SY5Y cells as well as in primary rat neurons. These findings suggest that DYRK1A protects against insulin resistance in the brain by elevating IRS-1 expression.

Four crustins involved in antibacterial responses in Marsupenaeus japonicus.

Crustins are a family of cationic, cysteine-rich antimicrobial peptides with a whey acidic protein (WAP) domain in the C-terminal. They have diverse functions in antimicrobial immune responses. Four groups of crustins (crustins I, II, III, and IV) have been identified in crustaceans, but type I crustins have not been reported in penaeid shrimp until now. In this study, we identified four crustins in kuruma shrimp Marsupenaeus japonicus, and named them MjCrus I-2, 3, 4 and 5. These four crustins belong to type I crustins, which contain a signal peptide, cysteine-rich region at the N-terminus, and WAP domain at the C-terminus. Tissue distribution demonstrated that MjCrus I-2, 3 and 5 had high expression levels in hemocytes, gills and stomach. whereas MjCrus I-4 was distributed in all tissues detected. MjCrus I-2 to 5 showed different expression patterns in different tissues after Gram-positive bacterial (Staphylococcus aureus), Gram-negative bacterial (Vibrio anguillarum), and white spot syndrome virus (WSSV) challenge. The expression of MjCrus I-2 to 5 was upregulated by bacterial or WSSV challenge. The three crustins were recombinantly expressed in Escherichia coli, and the purified proteins showed few antimicrobial activities. Three MjCrus Is could bind to different bacteria. MjCrus I-2 and 3 showed different inhibitory abilities to secreted bacterial proteases. MjCrus I-4 could not inhibit bacterial proteases. After knockdown of MjCrus I-3, the bacterial scavenging ability to V. anguillarum was impaired. These results suggested that type I crustins played an important role in the innate immunity of shrimp.

A novel crustin from Marsupenaeus japonicus promotes hemocyte phagocytosis.

Crustins are cationic cysteine-rich antimicrobial peptides (AMPs) that contain multiple domains (glycine-rich, cysteine-rich, or proline-rich) at the N-terminus and whey acidic protein (WAP) domains at the C-terminus. Crustins have multiple functions, including protease inhibition and antimicrobial activity. Other functions of crustins need to be clarified. In this study, a novel crustin with a cysteine-rich region, and a single WAP domain, belonging to type I crustins, was identified in Marsupenaeus japonicus and designated as MjCru I-1. MjCru I-1 was expressed in various tissues. The expression of MjCru I-1 was upregulated in the hemocytes of shrimp challenged with bacteria. MjCru I-1 could bind to bacteria by binding to the cell wall molecules of the bacteria, such as lipopolysaccharide (LPS), peptidoglycan (PGN), and lipoteichoic acid (LTA). The synthesized WAP domain of MjCru I-1 but not synthesized Cys-rich domain has antibacterial and agglutinative activities. Scanning electron microscope assay showed that the bacterial cells treated with sMjCru I-1 appeared to be disrupted and cracked compared with those of the control samples. The knockdown of MjCru I-1 could reduce bacterial clearance and injection of MjCru I-1 could significantly increase the survival rate of shrimp infected with Vibrio anguillarum and Staphylococcus aureus compared with those of the control samples. Further study discovered that MjCru I-1 could increase the hemocyte phagocytosis against V. anguillarum and S. aureus. These results suggest that MjCru I-1 has dual functions, bactericidal and phagocytosis promoting activities, in the antibacterial immunity of shrimp.

A new group of anti-lipopolysaccharide factors from Marsupenaeus japonicus functions in antibacterial response.

Anti-lipopolysaccharide factors (ALFs) are a group of critical effector molecules with a broad spectrum of antimicrobial activities in crustaceans. Four groups of ALFs (A, B, C, and D) have been identified in peneaid shrimp. In the study, we identified a new group of ALFs (designated as MjALF-E) from Marsupenaeus japonicus. This new group (group E) included MjALF-E1 and E2. MjALF-E1 was highly expressed in hemocytes, heart, and intestine, whereas E2 was highly expressed in gills, stomach, and intestine. Expressions of both MjALF-E1 and E2 were upregulated by bacterial challenge. Synthesized LPS-binding domain peptides of MjALF-E1 and E2 strongly bind to bacterial cell wall components lipopolysaccharide (LPS) and peptidoglycan (PGN). The recombinant rMjALF-E2 showed relatively weak binding activity to LPS and PGN. Both synthesized peptides and rMjALF-E2 exhibited antimicrobial activity against Gram-negative bacteria, whereas rMjALF-E2 could promote the clearance of bacteria in vivo. After knockdown of MjALF-E2 and infection with Vibrio anguillarum, shrimp showed high and rapid mortality compared with GFPi shrimp. These results suggest that MjALF-Es serves a protective function against bacterial infection in shrimp.

Calnexin functions in antibacterial immunity of Marsupenaeus japonicus.

Calnexin (Cnx) is an endoplasmic reticulum membrane-bound lectin chaperone that comprises a dedicated maturation system with another lectin chaperone calreticulin (Crt). This maturation system is known as the Cnx/Crt cycle. The main functions of Cnx are Ca(2+) storage, glycoprotein folding, and quality control of synthesis. Recent studies have shown that Cnx is important in phagocytosis and in optimizing dendritic cell immunity. However, the functions of Cnx in invertebrate innate immunity remain unclear. In this research, we characterized Cnx in the kuruma shrimp Marsupenaeus japonicus (designated as MjCnx) and detected its function in shrimp immunity. The expression of MjCnx was upregulated in several tissues challenged with Vibrio anguillarum. Recombinant MjCnx could bind to bacteria by binding polysaccharides. MjCnx protein existed in the cytoplasm and on the membrane of hemocytes and was upregulated by bacterial challenge. The recombinant MjCnx enhanced the clearance of V. anguillarum in vivo, and the clearance effects were impaired after silencing MjCnx with RNA interference assay. Recombinant MjCnx promoted phagocytosis efficiency of hemocytes. These results suggest that MjCnx functions as one of the pattern recognition receptors and has crucial functions in shrimp antibacterial immunity.