KCNN4 Promotes the Stemness Potentials of Liver Cancer Stem Cells by Enhancing Glucose Metabolism.

The presence of liver cancer stem cells (LCSCs) is one of the reasons for the treatment failure of hepatocellular carcinoma (HCC). For LCSCs, one of their prominent features is metabolism plasticity, which depends on transporters and ion channels to exchange metabolites and ions. The K+ channel protein KCNN4 (Potassium Calcium-Activated Channel Subfamily N Member 4) has been reported to promote cell metabolism and malignant progression of HCCs, but its influence on LCSC stemness has remained unclear. Here, we demonstrated that KCNN4 was highly expressed in L-CSCs by RT-PCR and Western blot. Then, we illustrated that KCNN4 promoted the stemness of HC-C cells by CD133+CD44+ LCSC subpopulation ratio analysis, in vitro stemness transcription factor detection, and sphere formation assay, as well as in vivo orthotopic liver tumor formation and limiting dilution tumorigenesis assays. We also showed that KCNN4 enhanced the glucose metabolism in LCSCs by metabolic enzyme detections and seahorse analysis, and the KCNN4-promoted increase in LCSC ratios was abolished by glycolysis inhibitor 2-DG or OXPHOS inhibitor oligomycin. Collectively, our results suggested that KCNN4 promoted LCSC stemness via enhancing glucose metabolism, and that KCNN4 would be a potential molecular target for eliminating LCSCs in HCC.

Single cell molecular alterations reveal target cells and pathways of conditioned fear memory.

OBJECTIVES: Recent evidence indicates that hippocampus is important for conditioned fear memory (CFM). Though few studies consider the roles of various cell types' contribution to such a process, as well as the accompanying transcriptome changes during this process. The purpose of this study was to explore the transcriptional regulatory genes and the targeted cells that are altered by CFM reconsolidation. METHODS: A fear conditioning experiment was established on adult male C57 mice, after day 3 tone-cued CFM reconsolidation test, hippocampus cells were dissociated. Using single cell RNA sequencing (scRNA-seq) technique, alterations of transcriptional genes expression were detected and cell cluster analysis were performed and compared with those in sham group. RESULTS: Seven non-neuronal and eight neuronal cell clusters (including four known neurons and four newly identified neuronal subtypes) has been explored. Among them, CA subtype 1 has characteristic gene markers of Ttr and Ptgds, which is speculated to be the outcome of acute stress and promotes the production of CFM. The results of KEGG pathway enrichment indicate the differences in the expression of certain molecular protein functional subunits in long-term potentiation (LTP) pathway between two types of neurons (DG and CA1) and astrocytes, thus providing a new transcriptional perspective for the role of hippocampus in the CFM reconsolidation. More importantly, the correlation between the reconsolidation of CFM and neurodegenerative diseases-linked genes is substantiated by the results from cell-cell interactions and KEGG pathway enrichment. Further analysis shows that the reconsolidation of CFM inhibits the risk-factor genes App and ApoE in Alzheimer's Disease (AD) and activates the protective gene Lrp1. CONCLUSIONS: This study reports the transcriptional genes expression changes of hippocampal cells driven by CFM, which confirm the involvement of LTP pathway and suggest the possibility of CFM-like behavior in preventing AD. However, the current research is limited to normal C57 mice, and further studies on AD model mice are needed to prove this preliminary conclusion.

TFF3 promotes clonogenic survival of colorectal cancer cells through upregulation of EP4 via activation of STAT3.

Background: While growing evidence indicates the importance of TFF3 in cancer, the molecular mechanism of its action in cancer remains largely unknown. Clonogenic survival is a key ability for tumor cells, which is interpreted as a trait of cancer cells with tumor-initiating capabilities. We investigated the effect and the underlying mechanisms of TFF3 on the clonogenic survival of colorectal cancer (CRC) cells. Methods: Expression of TFF3 in CRC tissues and matched paracancerous tissues was determined by western blotting. Colony formation assays were performed to evaluate the clonogenic survival ability of CRC cells. PTGER4 mRNA expression was detected by quantitative polymerase chain reaction. PTGER4 promoter activity was determined by luciferase reporter assay. STAT3 nuclear localization was investigated using immunofluorescence staining. Expression of TFF3 and EP4 in CRC tissues was determined by immunohistochemistry. Results: TFF3 knockout led to decreased clonogenic survival of CRC cells, while overexpression of TFF3 resulted in the opposite effect. EP4 was found to be upregulated by TFF3 at both the mRNA and protein level. Moreover, EP4 antagonist abrogated TFF3-mediated clonogenic survival of CRC cells. PGE2 and EP4 agonist could restore the effect of TFF3 knockout on the clonogenic survival of CRC cells. Furthermore, TFF3 promoted STAT3 activation and nuclear localization. Activated STAT3 bound to PTGER4 promoter, the gene encoding for EP4, and facilitated PTGER4 transcription. Conclusions: TFF3 promotes clonogenic survival of CRC cells via upregulating EP4 expression.

The combination of NDUFS1 with CD4+ T cell infiltration predicts favorable prognosis in kidney renal clear cell carcinoma.

Background: Kidney renal clear cell carcinoma (KIRC) is an immunogenic tumor, and immune infiltrates are relevant to patients' therapeutic response and prognosis. NDUFS1, the core subunit of mitochondrial complex I, has been reported to be associated with KIRC patients' prognosis. However, the upstream regulator for NDUFS1 and their correlations with immune infiltration remain unclear. Methods: The expression of NDUFS genes in KIRC and their influences on patients' survival were investigated by UALCAN, ENCORI, Oncomine, TIMER as well as Kaplan-Meier Plotter. miRNAs regulating NDUFS1 were predicted and analyzed by TargetScan and ENCORI. The correlations between NDUFS1 expression and immune cell infiltration or gene marker sets of immune infiltrates were analyzed via TIMER. The overall survival in high/low NDUFS1 or hsa-miR-320b expressed KIRC patients with or without immune infiltrates were analyzed via Kaplan-Meier Plotter. The combined NDUFS1 expression and/or CD4+ T cell infiltration on KIRC patients' overall survival were validated by multiplexed immunofluorescence (mIF) staining in tissue microarray (TMA). Furthermore, the influences of NDUFS1 expression on the chemotaxis of CD4+ T cells to KIRC cells were performed by transwell migration assays. Results: We found that the low expression of NDUFS1 mRNA and protein in KIRC was correlated with unfavorable patients' survival and poor infiltration of CD4+ T cells. In patients with decreased CD4+ T cell infiltration whose pathological grade less than III, TMA mIF staining showed that low expression of NDUFS1 had significantly poor OS than that with high expression of NDUFS1 did. Furthermore, hsa-miR-320b, a possible negative regulator of NDUFS1, was highly expressed in KIRC. And, low NDUFS1 or high hsa-miR-320b consistently correlated to unfavorable outcomes in KIRC patients with decreased CD4+ T cell infiltration. In vitro, NDUFS1 overexpression significantly increased the chemotaxis of CD4+ T cell to KIRC cells. Conclusion: Together, NDUFS1, upregulated by decreased hsa-miR-320b expression in KIRC patients, might act as a biomarker for CD4+ T cell infiltration. And, the combination of NDUFS1 with CD4+ T cell infiltration predicts favorable prognosis in KIRC.

Multifunctional chitosan/grape seed extract/silver nanoparticle composite for food packaging application.

Food-borne fungi present significant hazards to food preservation and human health. Oxidation causes spoilage and the inedibility of the fruit. However, traditional packaging films without antimicrobial or antioxidant activities do not satisfy the active packaging requirements. Films with antimicrobial and antioxidant activities are urgently required. In this study, silver nanoparticles (AgNPs) were synthesized from fruit waste grape seed extracts (GSE). The antimicrobial and antioxidant activities of GSE-silver nanoparticles (GSE-AgNPs) and AgNPs (average size 20 nm) stabilized by polyvinyl pyrrolidone (PVP-AgNPs) were evaluated in vitro. The effect of chitosan (CS)-coated GSE-AgNPs and PVP-AgNPs on the postharvest quality of grape was studied during storage at 20 °C for 5 days. The results confirmed that grapes treated with CS and GSE-AgNPs showed significantly reduced decay percentage, weight loss, and maintained titratable acidity at high levels compared with those of untreated fruit and fruit treated with PVP-AgNPs. Moreover, CS and GSE-AgNPs significantly inhibited the total mold count during storage. Our results suggest that CS coating enriched with GSE-AgNPs has the potential to preserve the quality and extend the shelf life of grapes.

Microbial volatile organic compounds: Antifungal mechanisms, applications, and challenges.

The fungal decay of fresh fruits and vegetables annually generates substantial global economic losses. The utilization of conventional synthetic fungicides is damaging to the environment and human health. Recently, the biological control of post-harvest fruit and vegetable diseases via antagonistic microorganisms has become an attractive possible substitution for synthetic fungicides. Numerous studies have confirmed the potential of volatile organic compounds (VOCs) for post-harvest disease management. Moreover, VOC emission is a predominant antifungal mechanism of antagonistic microorganisms. As such, it is of great significance to discuss and explore the antifungal mechanisms of microbial VOCs for commercial application. This review summarizes the main sources of microbial VOCs in the post-harvest treatment and control of fruit and vegetable diseases. Recent advances in the elucidation of antifungal VOC mechanisms are emphasized, and the applications of VOCs produced from antagonistic microorganisms are described. Finally, the current prospects and challenges associated with microbial VOCs are considered.

Chimeric antigen receptor T cells targeting CD147 for non-small cell lung cancer therapy.

Non-small cell lung cancer (NSCLC) is a highly malignant tumor, with a significant mortality and morbidity. With the development of tumor immunotherapy, chimeric antigen receptor T cells (CART) gets increasingly attention and achieves prominent contributions in the treatment of hematologic malignancies. However, CART therapy for NSCLC proceeds slowly and further researches need to be investigated. In our study, we performed bioinformatics analysis to evaluate the significant role of CD147 in NSCLC. The expression level of CD147 was detected in human NSCLC cell lines and NSCLC tissues. Meanwhile, CD147-CART was constructed and identified. Cell cytotoxicity and cytokine secretion were performed to evaluate the efficacy of CD147-CART. We also constructed cell-derived xenograft (CDX) model and patient-derived xenograft (PDX) model, which was used to further investigate the safety and efficacy of CD147-CART in vivo. Our observations show that CD147 is a specific tumor antigen of NSCLC and plays an essential role in NSCLC progression, which can be used as a target for CART therapy in NSCLC. CD147-CART cells exhibit robust cytotoxicity and cytokine production in vitro, suggesting a strong anti-tumor activity against NSCLC tumor cells. Importantly, CD147-CART cells have strong anti-tumor activity against NSCLC cells in vivo in both CDX and PDX models and no adverse side effects. Our findings show that CD147-CART immunotherapy for NSCLC is safe and effective, which is an ideal and promising medical patch for treating NSCLC.

Identification of Genes Linking Natural Killer Cells to Apoptosis in Acute Myocardial Infarction and Ischemic Stroke.

Natural killer (NK) cells are a type of innate lymphoid cell that are involved in the progression of acute myocardial infarction and ischemic stroke. Although multiple forms of programmed cell death are known to play important roles in these diseases, the correlation between NK cells and apoptosis-related genes during acute myocardial infarction and ischemic stroke remains unclear. In this study, we explored the distinct patterns of NK cell infiltration and apoptosis during the pathological progression of acute myocardial infarction and ischemic stroke using mRNA expression microarrays from the Gene Expression Omnibus database. Since the abundance of NK cells correlated positively with apoptosis in both diseases, we further examined the correlation between NK cell abundance and the expression of apoptosis-related genes. Interestingly, APAF1 and IRAK3 expression correlated negatively with NK cell abundance in both acute myocardial infarction and ischemic stroke, whereas ATM, CAPN1, IL1B, IL1R1, PRKACA, PRKACB, and TNFRSF1A correlated negatively with NK cell abundance in acute myocardial infarction. Together, these findings suggest that these apoptosis-related genes may play important roles in the mechanisms underlying the patterns of NK cell abundance and apoptosis in acute myocardial infarction and ischemic stroke. Our study, therefore, provides novel insights for the further elucidation of the pathogenic mechanism of ischemic injury in both the heart and the brain, as well as potential useful therapeutic targets.

A four oxidative stress gene prognostic model and integrated immunity-analysis in pancreatic adenocarcinoma.

Background and aims: Pancreatic adenocarcinoma (PAAD) is highly aggressive and characterized by a poor prognosis. Oxidative stress has great impacts on the occurrence and development of tumors. However, the predictive role of oxidative stress related genes on PAAD patients' prognosis remains unclear. In this study, we aimed to construct a prognostic model for PAAD based on oxidative stress genes and to evaluate its predictive value. Methods: The Cancer Genome Atlas (TCGA) and three Gene Expression Omnibus (GEO) datasets were used to identify differentially expressed oxidative stress genes. Univariate Cox regression, Kaplan-Meier and multivariate Cox regression analysis were used to select genes and to construct a prognosis model. According to the median value of the model's risk score, patients were divided into high and low risk groups, and gene set enrichment analysis (GSEA), immune infiltration and immunotherapy effect, drug resistance and the expression of immune checkpoint related genes and synthetic driver genes of T cell proliferation were analyzed. Finally, the mRNA and protein levels of four genes in PAAD were verified by the clinical proteomic tumor analysis consortium (CPTAC) database and the immunostaining of patients' tissue. Results: 55 differentially expressed oxidative stress genes were identified, and four genes including MET, FYN, CTTN and CDK1 were selected to construct a prognosis model. GESA indicated that immune related pathways, metabolic pathways and DNA repair pathways were significantly enriched in the high risk group as compared to the low risk group. The frequency of genetic mutations was also significantly higher in high risk groups than that in low risk groups. Moreover, the infiltration level of 23 immune cells as well as the expression of immune checkpoint related and synthetic driver genes of T cell proliferation were significantly altered, with the better immunotherapy effect occurring in low risk group. In patient PAAD tissues, the mRNA and protein levels of these four genes were up-regulated. Conclusion: We have successfully constructed a four oxidative stress gene prognostic model that has important predictive value for PAAD patients, and this model might be a promising guidance for prognostic prediction and efficacy monitoring in clinical individualized therapy.

PDGFA-associated protein 1 is a novel target of c-Myc and contributes to colorectal cancer initiation and progression.

BACKGROUND: The mechanism underlying colorectal cancer (CRC) initiation and progression remains elusive, and overall survival is far from satisfactory. Previous studies have shown that PDGFA-associated protein 1 (PDAP1) is upregulated in several cancers including CRC. Here, we aimed to identify the cause and consequence of PDAP1 dysregulation in CRC and evaluate its role as a potential therapeutic target. METHODS: Multi-omics data analysis was performed to identify potential key players in CRC initiation and progression. Immunohistochemistry (IHC) staining was applied to determine the expression pattern of PDAP1 in CRC tissues. Pdap1 conditional knockout mice were used to establish colitis and CRC mouse models. RNA sequencing, a phosphoprotein antibody array, western blotting, histological analysis, 5-bromo-2'-deoxyuridine (BrdU) incorporation assay, and interactome analysis were applied to identify the underlying mechanisms of PDAP1. A human patient-derived xenograft (PDX) model was used to assess the potential of PDAP1 as a therapeutic target. RESULTS: PDAP1 was identified as a potential key player in CRC development using multi-omics data analysis. PDAP1 was overexpressed in CRC cells and correlated with reduced overall survival. Further investigation showed that PDAP1 was critical for the regulation of cell proliferation, migration, invasion, and metastasis. Significantly, depletion of Pdap1 in intestinal epithelial cells impaired mucosal restitution in dextran sulfate sodium salt-induced colitis and inhibited tumor initiation and growth in colitis-associated cancers. Mechanistic studies showed that c-Myc directly transactivated PDAP1, which contributed to the high PDAP1 expression in CRC cells. PDAP1 interacted with the juxtamembrane domain of epidermal growth factor receptor (EGFR) and facilitated EGFR-mitogen-activated protein kinase (MAPK) signaling activation, which resulted in FOS-related antigen 1 (FRA-1) expression, thereby facilitating CRC progression. Notably, silencing of PDAP1 could hinder the growth of patient-derived xenografts that sustain high PDAP1 levels. CONCLUSIONS: PDAP1 facilitates mucosal restitution and carcinogenesis in colitis-associated cancer. c-Myc-driven upregulation of PDAP1 promotes proliferation, migration, invasion, and metastasis of CRC cells via the EGFR-MAPK-FRA-1 signaling axis. These findings indicated that PDAP1 inhibition is warranted for CRC patients with PDAP1 overexpression.

CD147 supports paclitaxel resistance via interacting with RanBP1.

Though the great success of paclitaxel, the variable response of patients to the drug limits its clinical utility and the precise mechanisms underlying the variable response to paclitaxel remain largely unknown. This study aims to verify the role and the underlying mechanisms of CD147 in paclitaxel resistance. Immunostaining was used to analyze human non-small-cell lung cancer (NSCLC) and ovarian cancer tissues. RNA-sequencing was used to identify downstream effectors. Annexin V-FITC/propidium iodide and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining were used to detect apoptosis. Co-immunoprecipitation (Co-IP), fluorescence resonance energy transfer (FRET) and surface plasmon resonance (SPR) were performed to determine protein interactions. Fluorescence recovery after photobleaching (FRAP) was performed to measure the speed of microtubule turnover. Xenograft tumor model was established to evaluate sensitivity of cancer cells to paclitaxel in vivo. In vitro and in vivo assays showed that silencing CD147 sensitized the cancer cells to paclitaxel treatment. CD147 protected cancer cells from paclitaxel-induced caspase-3 mediated apoptosis regardless of p53 status. Truncation analysis showed that the intracellular domain of CD147 (CD147ICD) was indispensable for CD147-regulated sensitivity to paclitaxel. Via screening the interacting proteins of CD147ICD, Ran binding protein 1 (RanBP1) was identified to interact with CD147ICD via its C-terminal tail. Furthermore, we showed that RanBP1 mediated CD147-regulated microtubule stability and dynamics as well as response to paclitaxel treatment. These results demonstrated that CD147 regulated paclitaxel response by interacting with the C-terminal tail of RanBP1 and targeting CD147 may be a promising strategy for preventing paclitaxel resistant.

CD147 receptor is essential for TFF3-mediated signaling regulating colorectal cancer progression.

Major gaps in understanding the molecular mechanisms of colorectal cancer (CRC) progression and intestinal mucosal repair have hampered therapeutic development for gastrointestinal disorders. Trefoil factor 3 (TFF3) has been reported to be involved in CRC progression and intestinal mucosal repair; however, how TFF3 drives tumors to become more aggressive or metastatic and how TFF3 promotes intestinal mucosal repair are still poorly understood. Here, we found that the upregulated TFF3 in CRC predicted a worse overall survival rate. TFF3 deficiency impaired mucosal restitution and adenocarcinogenesis. CD147, a membrane protein, was identified as a binding partner for TFF3. Via binding to CD147, TFF3 enhanced CD147-CD44s interaction, resulting in signal transducer and activator of transcription 3 (STAT3) activation and prostaglandin G/H synthase 2 (PTGS2) expression, which were indispensable for TFF3-induced migration, proliferation, and invasion. PTGS2-derived PGE2 bound to prostaglandin E2 receptor EP4 subtype (PTGER4) and contributed to TFF3-stimulated CRC progression. Solution NMR studies of the TFF3-CD147 interaction revealed the key residues critical for TFF3 binding and the induction of PTGS2 expression. The ability of TFF3 to enhance mucosal restitution was weakened by a PTGS2 inhibitor. Blockade of TFF3-CD147 signaling using competitive inhibitory antibodies or a PTGS2 inhibitor reduced CRC lung metastasis in mice. Our findings bring strong evidence that CD147 is a novel receptor for TFF3 and PTGS2 signaling is critical for TFF3-induced mucosal restitution and CRC progression, which widens and deepens the understanding of the molecular function of trefoil factors.