TMErisk score: A tumor microenvironment-based model for predicting prognosis and immunotherapy in patients with head and neck squamous cell carcinoma.

Tumor microenvironment (TME) is closely associated with the progression and prognosis of head and neck squamous cell carcinoma (HNSCC). To investigate potential biomarkers for predicting therapeutic outcomes in HNSCC, we analyzed the immune and stromal status of HNSCC based on the genes associated with TME using the ESTIMATE algorithm. Immune and stromal genes were identified with differential gene expression and weighted gene co-expression network analysis (WGCNA). From these genes, 118 were initially selected through Cox univariate regression and then further input into least absolute shrinkage and selection operator (LASSO) regression analysis. As a result, 11 genes were screened out for the TME-related risk (TMErisk) score model which presented promising overall survival predictive potential. The TMErisk score was negatively associated with immune and stromal scores but positively associated with tumor purity. Individuals with high TMErisk scores exhibited decreased expression of most immune checkpoints and all human leukocyte antigen family genes, and reduced abundance of infiltrating immune cells. Divergent genes were mutated in HNSCC. In both high and low TMErisk score groups, the tumor protein P53 exhibited the highest mutation frequency. A higher TMErisk score was found to be associated with reduced overall survival probability and worse outcomes of immunotherapy. Therefore, the TMErisk score could serve as a valuable model for the outcome prediction of HNSCC in clinic.

Altered intestinal microbiota enhances adenoid hypertrophy by disrupting the immune balance.

Introduction: Adenoid hypertrophy (AH) is a common upper respiratory disorder in children. Disturbances of gut microbiota have been implicated in AH. However, the interplay of alteration of gut microbiome and enlarged adenoids remains elusive. Methods: 119 AH children and 100 healthy controls were recruited, and microbiome profiling of fecal samples in participants was performed using 16S rRNA gene sequencing. Fecal microbiome transplantation (FMT) was conducted to verify the effects of gut microbiota on immune response in mice. Results: In AH individuals, only a slight decrease of diversity in bacterial community was found, while significant changes of microbial composition were observed between these two groups. Compared with HCs, decreased abundances of Akkermansia, Oscillospiraceae and Eubacterium coprostanoligenes genera and increased abundances of Bacteroides, Faecalibacterium, Ruminococcus gnavus genera were revealed in AH patients. The abundance of Bacteroides remained stable with age in AH children. Notably, a microbial marker panel of 8 OTUs were identified, which discriminated AH from HC individuals with an area under the curve (AUC) of 0.9851 in the discovery set, and verified in the geographically different validation set, achieving an AUC of 0.9782. Furthermore, transfer of mice with fecal microbiota from AH patients dramatically reduced the proportion of Treg subsets within peripheral blood and nasal-associated lymphoid tissue (NALT) and promoted the expansion of Th2 cells in NALT. Conclusion: These findings highlight the effect of the altered gut microbiota in the AH pathogenesis.

Curcumin Synergistically Enhances the Cytotoxicity of Arsenic Trioxide in U266 Cells by Increasing Arsenic Uptake.

Despite the constant emergence of new methods for the treatment of multiple myeloma (MM), relapse and drug resistance still exist, especially in MM with p53 mutations. Arsenic trioxide (ATO) can be used in MM treatment, but this single drug has poor effectiveness and also side effects. Curcumin is a safe and effective compound that can enhance the anticancer effects of many drugs. Previous studies have suggested that tumor cell sensitivity to ATO is related to the intracellular arsenic content, and aquaporin 9 (AQP9) is the key factor that determines intracellular arsenic content. This study aimed to explore whether curcumin can increase ATO cytotoxicity in MM and whether the mechanism is related to the regulation of intracellular arsenic content. U266 was treated with ATO, curcumin, and their combination, and cell proliferation, apoptosis, and intracellular arsenic content were detected by CCK-8 assay, flow cytometry, and HPLC-ICP-MS, respectively. AQP9 mRNA and protein levels were detected by qPCR and western blotting. The levels of Mcl-1, Bcl-2, Bax, caspase-3, and cleaved caspase-3 protein were detected by western blotting. ATO-induced cytotoxicity to U266 occurred in a time- and dose-dependent manner, but the therapeutic efficacy at low drug concentrations was modest. The arsenic content in U266 was lower than that in NB4, and the arsenic uptake by U266 was concentration-dependent. The expression levels of AQP9 mRNA and AQP9 protein in U266 were lower than those in NB4. Curcumin significantly enhanced the lethality of ATO to U266. The arsenic content in U266 in the combined drug group increased significantly compared with ATO treatment alone. After curcumin treatment, the AQP9 mRNA and AQP9 protein expression levels in U266 also increased. Compared with the control group, the expression of antiapoptotic proteins Mcl-1 and Bcl-2 decreased, the expression of proapoptotic protein Bax increased, the ratio of Bax/Bcl-2 increased, and the expression of caspase-3 decreased and cleaved caspase-3 increased in the combined drug groups. Curcumin can enhance the killing effects of ATO on U266 by increasing the intracellular arsenic content, which may be related to the upregulation of AQP9 expression. The combination of these two drugs is expected to be a potential clinical treatment for MM.

An Evaluation of Morphological Changes and Deformability of Suspended Red Blood Cells Prepared Using Whole Blood with Different Hemoglobin Levels of Tibetans.

BACKGROUND: The hypoxic environment stimulates the human body to increase the levels of hemoglobin (HGB) and hematocrit and the number of red blood cells. Such enhancements have individual differences, leading to a wide range of HGB in Tibetans' whole blood (WB). STUDY DESIGN: WB of male Tibetans was divided into 3 groups according to different HGB (i.e., A: >120 but ≤185 g/L, B: >185 but ≤210 g/L, and C: >210 g/L). Suspended red blood cells (SRBC) processed by collected WB and stored in standard conditions were examined aseptically on days 1, 14, 21, and 35 after storage. The routine biochemical indexes, deformability, cell morphology, and membrane proteins were tested. RESULTS: Mean corpuscular volume, adenosine triphosphate, pH, and deformability were not different in group A vs. those in storage (p > 0.05). The increased rate of irreversible morphology of red blood cells was different among the 3 groups, but there was no difference in the percentage of red blood cells with an irreversible morphology after 35 days of storage. Group C performed better in terms of osmotic fragility and showed a lower rigid index than group A. Furthermore, SDS-PAGE revealed similar cross-linking degrees of cell membrane protein but the band 3 protein of group C seemed to experience weaker clustering than that of group A as detected by Western Blot analysis after 35 days of storage. CONCLUSIONS: There was no difference in deformability or morphological changes in the 3 groups over the 35 days of storage. High HGB levels of plateau SRBC did not accelerate the RBC change from a biconcave disc into a spherical shape and it did not cause a reduction in deformability during 35 days of preservation in bank conditions.

Upregulation of Nogo-B by hypoxia inducible factor-1 and activator protein-1 in hepatocellular carcinoma.

Nogo-B is an important regulator of tumor angiogenesis. Expression of Nogo-B is remarkably upregulated in multiple tumor types, especially hepatocellular carcinoma (HCC). Here, we show the transcriptional regulation mechanisms of Nogo-B in liver cancer. In response to hypoxia, expression of Nogo-B significantly increased in HCC tissues and cells. The distal hypoxia-responsive element in the promoter was essential for transcriptional activation of Nogo-B under hypoxic conditions, which is the specific site for hypoxia inducible factor-1α (HIF-1α) binding. In addition, Nogo-B expression was associated with c-Fos expression in HCC tissues. Nogo-B expression was induced by c-Fos, yet inhibited by a dominant negative mutant A-Fos. Deletion and mutation analysis of the predicted activator protein-1 binding sites revealed that functional element mediated the induction of Nogo-B promoter activity, which was confirmed by ChIP. These results indicate that HIF-1α and c-Fos induce the expression of Nogo-B depending on tumor microenvironments, such as hypoxia and low levels of nutrients, and play a role in upregulation of Nogo-B in tumor angiogenesis.

Bi-allelic mutation in Fsip1 impairs acrosome vesicle formation and attenuates flagellogenesis in mice.

Fibrous sheath interacting protein 1 (Fsip1) is a cytoskeletal structural protein of the sperm flagellar proteome. A few studies have reported that it plays a vital role in the tumorigenesis and cancer progression. However, little is known about the role of Fsip1 in spermatogenesis and mammalian sperm flagellogenesis. Fsip1 protein showed the highest expression in round spermatids, and was translocated from nucleus to the anterior region of the elongating spermatid head. To investigate its role we constructed homozygous Fsip1 null (Fsip1-/-) mice. We found that the homozygous Fsip1-/- mutant mice were infertile, with a low sperm count and impaired motility. Interestingly, a subtle phenotype characterized by abnormal head shape, and flagella deformities was observed in the sperm of Fsip1-/- mutant mice similar to the partial globozoospermia phenotype. Electron microscopy analysis of Fsip1-/- sperm revealed abnormal accumulation of mitochondria, disrupted axoneme and retained cytoplasm. Testicular sections showed increased cytoplasmic vacuoles in the elongated spermatid of Fsip1-/-mice, which indicated an intraflagellar transport (IFT) defect. Using proteomic approaches, we characterized the cellular components and the mechanism underlying this subtle phenotype. Our result indicated that Fsip1-/-downregulates the formation of acrosomal membrane and vesicles proteins, intraflagellar transport particles B, and sperm flagellum components. Our results suggest that Fsip1 is essential for normal spermiogenesis, and plays an essential role in the acrosome biogenesis and flagellogenesis by attenuating intraflagellar transport proteins.

[Relationship between Iron Metabolism and Platelet Parameters in Patients with Iron Deficiency Anemia].

OBJECTIVE: To investigate the relationship between iron metabolism and thrombocytosis. METHODS: iron metabolism indexes , erythrocyte and platelet parameters of iron deficiency anemia patients with thrombocytosis were collected, the correlation of platelet parameters with iron metabolism indexes and erythrocyte parameters was analysed; the difference in erythrocyte，platelet and iron parameters between severe anemia group (hemoglobin<60 g / L) and mild and moderate anemia group (hemoglobin≥60 g / L) were compared; the changes of platelet count before and after treatment were observed. RESULTS: There was a significant negative correlation between serum iron and platelet count (r=-0.404,P<0.01).Serum iron negatively correlated with platelet crit(r=-0.288,P<0.05). Transferrin saturation negatively correlated with platelet count and platelet crit(r=-0.353,P<0.01;r=-0.271, P<0.05).Serum ferritin and total iron binding capacity revealed no significant relation with any platelet parameters.Hemoglobin level , hematocrit and mean corpuscular hemoglobin concentration negatively correlated with platelet count(r=-0.239,P<0.05;r=-0.250,P<0.05;r=-0.339,P<0.01).There were differences in iron metabolism indexes and platelet parameters between mild to moderate anemia group and severe anemia group. After treatment, the platelet count decreased or was normal. CONCLUSION: The important iron metabolism indexes affecting platelet count are serum iron and transferrin saturation,that is,the severer iron deficiency, the higher platelet count.Patients with more severe and hypochronic anemia has higher platelet count.

Nogo-B promotes tumor angiogenesis and provides a potential therapeutic target in hepatocellular carcinoma.

Tumor angiogenesis is one of the hallmarks of cancer as well as an attractive target for cancer therapy. Characterization of novel pathways that act in parallel with the VEGF/VEGFR axis to promote tumor angiogenesis may provide insights into novel anti-angiogenic therapeutic targets. We found that the expression level of Nogo-B is positively correlated with tumor vessel density in hepatocellular carcinoma (HCC). While Nogo-B depletion inhibited tumor angiogenesis, Nogo-B overexpression promoted tumor angiogenesis in a tumor xenograft subcutaneous model of the human HCC cell line. Mechanically, Nogo-B regulates tumor angiogenesis based on its association with integrin αv ß3 and activation of focal adhesion kinase. Moreover, Nogo-B antibody successfully abolished the function of Nogo-B in tumor angiogenesis in vitro and in vivo. Collectively, our results strongly suggest that Nogo-B is an important tumor angiogenic factor and blocking Nogo-B selectively inhibits tumor angiogenesis.

TCP10L acts as a tumor suppressor by inhibiting cell proliferation in hepatocellular carcinoma.

TCP10L (T-complex 10 (mouse)-like) has been identified as a liver and testis-specific gene. Although a potential transcriptional suppression function of TCP10L has been reported previously, biological function of this gene still remains largely elusive. In this study, we reported for the first time that TCP10L was significantly down-regulated in clinical hepatocellular carcinoma (HCC) samples when compared to the corresponding non-tumorous liver tissues. Furthermore, TCP10L expression was highly correlated with advanced cases exceeding the Milan criteria. Overexpression of TCP10L in HCC cells suppressed colony formation, inhibited cell cycle progression through G0/G1 phase, and attenuated cell growth in vivo. Consistently, silencing of TCP10L promoted cell cycle progression and cell growth. Therefore, our study has revealed a novel suppressor role of TCP10L in HCC, by inhibiting proliferation of HCC cells, which may facilitate the diagnosis and molecular therapy in HCC.

p53 Represses transcription of RING finger LIM domain-binding protein RLIM through Sp1.

RLIM acts as a negative regulator of LIM-Homeodomain proteins either by recruiting Sin3A/Histone Deacetylase (HDAC) co-repressor complex or through degradation of CLIM coactivator, thus playing an important role in embryonic development. Recent studies by different research groups have shown that RLIM acts as an X-encoded, dose-dependent inducer of X chromosome inactivation in mouse embryonic stem cells. However, until now, very little is known about the expression regulation of RLIM gene, and we tried to study the transcriptional regulation of RLIM gene. In the present study, we identified RLIM as a novel target of p53 and demonstrated that p53 repressed both mRNA and protein levels of RLIM. Expression of wild type p53, but not p53 mutants, led to repression of the RLIM promoter activity. We further identified four putative Sp1 elements (S1 to S4) on the RLIM promoter that are essential for p53-mediated repression of RLIM. Although p53 does not directly bind to the RLIM promoter, it physically interacts with and prevents the binding of Sp1 to the RLIM promoter. Thus, RLIM is a novel target of p53, and p53 exerts its inhibitory effect on RLIM expression by interfering with Sp1-mediated transcriptional activation on RLIM. Our results provided data to enlarge the knowledge of transcriptional regulation of RLIM and suggested a new pathway by which physiological and pathological activators of p53 may affect development.

DNAJC25 is downregulated in hepatocellular carcinoma and is a novel tumor suppressor gene.

HSP40, also known as DnaJ, is one of the subfamilies of the heat shock protein family. DnaJ/Hsp40 proteins act as co-chaperones by binding to the chaperone Hsp70 through their J domain and stimulating ATP hydrolysis to aid protein translation, folding, unfolding, translocation and degradation. They are implicated in various human diseases, including neurodegenerative disorders and cancer. In the present study, we cloned and identified a new gene, DnaJ (HSP40) homolog, subfamily C, member 25 (DNAJC25), which is localized to the cytoplasm. Real-time PCR revealed that the expression of DNAJC25 is particularly high in the liver and is down-regulated in hepatocellular carcinoma (HCC) compared with adjacent normal tissues. The overexpression of DNAJC25 led to an inhibition of colony growth both in quantity and size. Flow cytometry analysis indicated that DNAJC25 also significantly increased cell apoptosis. Our data, therefore, indicate that DNAJC25 plays an important role in hepatocellular carcinogenesis, and should be further studied as a potential tumor suppressor candidate.

Metallothionein MT1M is a tumor suppressor of human hepatocellular carcinomas.

Members of the metallothionein (MT) family are short, cysteine-rich proteins involved in metal metabolism and detoxification, suggesting that MT proteins protect cells from damage caused by electrophilic carcinogens and thereby constitute a critical surveillance system against carcinogenesis. However, the roles of MT proteins in human hepatocellular carcinoma (HCC) are not fully understood. We identified a member of the MT family, termed MT1M. MT1M is expressed in various normal tissues with the highest level in the liver. MT1M expression can be induced by heavy metals and protect Escherichia coli from heavy metal toxicity. However, MT1M expression markedly decreased in human HCC specimens. A methylation profiling analysis indicated that the MT1M promoter is methylated in the majority of HCC tumors examined. Moreover, restored expression of MT1M in the HCC cell line Hep3B, which lacks endogenous MT1M expression, suppressed cell growth in vitro and in vivo and augmented apoptosis induced by tumor necrosis factor α. Furthermore, stable expression of MT1M in Hep3B cells blocked tumor necrosis factor α-induced degradation of IκBα and transactivation of NF-κB. We conclude that MT1M is a novel member of the MT family. Frequent downregulation of MT1M in human HCC may contribute to liver tumorigenesis by increasing cellular NF-κB activity.

Zinc finger transcription factor 191, directly binding to ß-catenin promoter, promotes cell proliferation of hepatocellular carcinoma.

UNLABELLED: Activation of ß-catenin, the central effector of the canonical wingless-type (Wnt) pathway, has been implicated in hepatocellular carcinoma (HCC). However, the transcription regulation mechanism of the ß-catenin gene in HCC remains unknown. Here we report that human zinc finger protein 191 (ZNF191) is a potential regulator of ß-catenin transcription. ZNF191, a Krüppel-like protein, specifically interacts with the TCAT motif, which constitutes the HUMTH01 microsatellite in the tyrosine hydroxylase (TH) gene ex vivo. We demonstrate that ZNF191 is significantly overexpressed in human HCC specimens and is associated with growth of human HCC cells. Global profiling of gene expression in ZNF191 knockdown human hepatic L02 cells revealed that the important Wnt signal pathway genes ß-catenin and cyclin D1 messenger RNAs (mRNAs) are significantly down-regulated. In agreement with transcription level, ß-catenin and cyclin D1 proteins are also down-regulated in transient and stable ZNF191 knockdown L02 and hepatoma Hep3B cell lines. Moreover, significant correlation between ZNF191 and ß-catenin mRNA expression was detected in human HCCs. Promoter luciferase assay indicated that ZNF191 can increase transcription activity of the full-length ß-catenin (CTNNB1) promoter, and nucleotide (nt)-1407/-907 of the CTNNB1 promoter exhibited the maximum transcriptional activity. Electrophoretic mobility shift assay showed that purified ZNF191 protein can directly bind to the CTNNB1 promoter, and the binding region is located at nt-1254/-1224. Finally, we demonstrate that the key binding sequence of ZNF191 in vivo is ATTAATT. CONCLUSION: ZNF191 can directly bind to the CTNNB1 promoter and activate the expression of ß-catenin and its downstream target genes such as cyclin D1 in hepatoma cell lines. This study uncovers a new molecular mechanism of transcription regulation of the ß-catenin gene in HCC.

Characterization of neuritin as a novel angiogenic factor.

Neuritin (NRN1), a neurotrophic factor, plays an important role in neurite growth and neuronal survival. In this study, we identify a new function of neuritin as a novel angiogenic factor in vitro and in vivo. Recombinant neuritin protein had no effect on the proliferation and adhesion of human umbilical vein endothelial cells (HUVEC), but it dose-dependently increased endothelial cell migration. Furthermore, overexpression of neuritin significantly promoted tumor angiogenesis, and surprisingly, it inhibited tumor growth in a xenograft tumor model. Thus, our results indicate that neuritin may act as an important angiogenic factor and serve as a potential target for cancer therapy.