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Patients with primary central nervous system lymphoma (PCNSL) have a prognosis poorer than that of systemic lymphoma patients. In patients with this condition, TLR4/STAT3 pathway alterations and the MYD88 L265P mutation may be viable targets for therapeutic intervention. The present study was, therefore, designed to identify clinicopathologic correlates of MYD88 mutations and TLR4/STAT3 pathway alterations in PCNSL. We detected TLR4 and p-STAT3 in 41.5% (22/53) and 43.4% (23/53) of PCNSL patients, respectively, while 60.4% of these patients (32/53) were found to harbor the MYD88 L265P mutation. TLR4 expression was found to be significantly associated with the presence of multiple brain lesions, while p-STAT3 expression was significantly linked to advanced age, the presence of multiple brain lesions, non-GCB histological findings, and non-CR status. The presence of the MYD88 L265P mutation was significantly linked to advanced age, the presence of multiple brain lesions, and DLBCL molecular subtype. Multivariate analyses additionally confirmed that elevated TLR4 and p-STAT3 expression levels are associated with a poorer PCNSL patient prognosis. Based on these findings, we hypothesize that signaling through the TLR4/MYD88/STAT3 pathway plays a key role in the pathogenesis of PCNSL.

Recently we found that ATP5J was over-expressed in tissue samples from patients with colorectal cancer. However, the clinical significance and function of the over-expression of ATP5J in these patients remains unclear. We investigated these issues in the current study. Our results indicated that expression of ATP5J was significantly higher in colorectal cancer tissue than in adjacent tissue, and it was also significantly higher in metastatic lymph nodes than in primary cancer tissue (P<0.05). A correlation between ATP5J expression and tumor differentiation was detected, but no correlation with gender, age, T stage, lymph node metastasis, or survival status was observed. Down-regulation of ATP5J expression attenuated the ability of cell migration and increased the sensitivity to 5-fluorouracil (5-Fu) in cells of the DLD1 cell line. Inversely, up-regulation of ATP5J expression enhanced cell migration and decreased 5-Fu sensitivity, suggesting that the function of ATP5J in colorectal cancer might involve cell migration and 5-Fu sensitivity.

This study aimed to elucidate the differences in vulval squamous cell carcinomas (VSCC) based on the HPV infection status. The sequencing data GSE183454 which contains 23 VSCC samples based on its HPV infection status was obtained from the Gene Expression Omnibus (GEO) database. We comprehensively dissected the differences of genomic and tumour microenvironment (TME) immune cell infiltration landscapes between HPV + and HPV- VSCC. The potential molecular mechanisms of prognostic genes were explored by functional enrichment analysis. Five novel key molecules (SYCP2, SMC1B, RNF212, MAJIN and C14orf39) with significantly up-regulated expression in HPV + VSCC were identified while protein-protein interaction (PPI) networks were created upon Cytoscape software. Additionally, VSCC with up-regulated expression of these key molecules exhibited a significantly decreased TME immune cell infiltration. SYCP2 is overexpressed in HPV + VSCC and could be a candidate therapy target for further research. What is already known on this subject? VSCC are characterised by two aetiological pathways. The former occurs in the background of lichen sclerosus, while the latter is related to HPV infection. VSCC most commonly arises from the non-HPV related pathway portends worse prognosis than VSCC derived from HPV infection. What do the results of this study add? Five key molecules are identified and significantly up-regulated in HPV + VSCC. In which, SYCP2 is overexpressed in HPV + VSCC and exhibited a significantly decreased TME immune cell infiltration. SYCP2 constant expression could be a potential biomarker of neoplasms associated with HPV and could be a candidate therapy target in VSCC especially HPV + VSCC for further research. What are the implications of these findings for clinical practice and/or further research? SYCP2 could be a candidate therapy target in VSCC especially with HPV + for further research. [ABSTRACT FROM AUTHOR]

Alkaline mineral complex buffer can improve rumen fermentation and affect the rumen microbiota of dairy cows. Here, we studied the effects of alkaline mineral complex buffer on serum immunity indexes, rumen fermentation and the microbiota of newborn calves. We also investigated changes in the rumen epithelial transcriptome expression profile. Compared with the control group, at 15 d, the serum contents of TP and GLB in the treatment group increased significantly (p < 0.05). At 30 d, the serum contents of GLB in the treatment group increased significantly (p < 0.05). At 45 d, the serum contents of IgG in the treatment group increased significantly (p < 0.05). At 60 d, the serum contents of TP and IgG in the treatment group increased significantly (p < 0.05). Rumen pH in the treatment groups was significantly increased at different days of age (p < 0.05). The microbial community composition in the rumen was determined using bacterial and archaeal 16S ribosomal RNA (rRNA) gene amplicon-sequencing. Analysis of bacterial composition in the rumen showed that there was no significant difference in bacterial diversity (p > 0.05). At the phylum level, Firmicutes were significantly decreased and Bacteroidetes were significantly increased in the treatment group at 30 d (p < 0.05). At the genus level, Prevotella_1, Olsenella, Christensenellaceae_R-7_group were significantly increased, and Lachnospiraceae_NK3A20_group, Ruminococcaceae_UCG-014 and Ruminococcus_2 were significantly decreased in the treatment group at 30 d (p < 0.05). Christensenellaceae_R-7_group was significantly increased in the treatment group (p < 0.05) at 45 d. Prevotella_9 was significantly decreased, and Prevotellaceae_UCG_001, Christensenellaceae_R-7_group were significantly increased in the treatment group at 60 d (p < 0.05). RNA sequence analysis of the rumen epithelium showed that 232 differentially expressed genes were screened, of which 158 were upregulated and 74 were downregulated. The main enrichment pathway was related to immune regulation. In conclusion, alkaline mineral complex buffer can enhance the body's immune response, regulate rumen fermentation by regulating the abundance of rumen microbiota and upregulate immune-related genes in rumen tissues to promote immune regulation. The results of this study provide a reference for the early nutritional regulation of newborn calves with an alkaline mineral complex buffer. [ABSTRACT FROM AUTHOR]

Background: Cancer metastasis is still a life threat to patients with colorectal cancer (CRC). Brain and muscle ARNT‐like protein 1 (BMAL1) is an important biological proteins that can regulate the behavior of cancer cells and their response to chemotherapy. However, the role of BMAL1 in the tumorigenic phenotype of CRC remains unclear. Here, we aim to investigate the functional role and mechanisms of BMAL1 in CRC. Methods: The mRNA expression of BMAL1 was studied using the Cancer Genome Atlas (TCGA) databases. The protein level in clinical tissues was confirmed by immunohistochemistry (IHC). The effects of BMAL1 on the epithelial‐to‐mesenchymal transition (EMT) and proliferation of CRC cell lines (including BMAL1 overexpressed or silencing cells) were studied by Transwell, wound healing, CCK‐8 and colony formation experiments. A series of experiments were conducted to demonstrate the mechanisms of BMAL1 regulating EMT and cancer proliferation in vitro and in vivo. Results: We found that BMAL1 expression was closely related to the poor prognosis of CRC. BMAL1 overexpression promoted cell proliferation and migration. Mechanistically, we found that BMAL1 may activate the epithelial‐to‐mesenchymal transition (EMT) pathway and induce the β‐catenin release further promotes the expression of oncogene c‐Myc and the migration of colorectal cells by activating MAPK pathway. However, BMAL1 silencing achieved the opposite effect. In addition, blocking MAPK‐signaling pathway with specific inhibitors of ERK1/2 and JNK can also downregulate the expressions of c‐Myc in vitro. Taken together, these results suggested that the BMAL1/ c‐Myc‐signaling pathway may regulate the metastasis of CRC through the JNK/ERK1/2 MAPK‐dependent pathway. Conclusions: Our study showed that BMAL1 promotes CRC metastasis through MAPK‐c‐Myc pathway. These results deepen our understanding of the relationship between BMAL1 and tumorigenic phenotypes, which may become a promising therapeutic target for BMAL1 overexpressing CRC. [ABSTRACT FROM AUTHOR]

Saliva is a convenient non-invasive source of liquid biopsy to monitor human health and diagnose diseases. In particular, extracellular vesicles (EVs) in saliva can potentially reveal clinically relevant information for systemic health. Recent studies have shown that RNA in saliva EVs could be exploited as biomarkers for disease diagnosis. However, there is no standardized protocol for profiling RNA in saliva EV nor clear guideline on selecting saliva fractions for biomarker analysis. To address these issues, we established a robust protocol for small RNA profiling from fractionated saliva. With this method, we performed comprehensive small RNA sequencing of four saliva fractions, including cell-free saliva (CFS), EV-depleted saliva (EV-D), exosome (EXO), and microvesicle (MV) from ten healthy volunteers. By comparing the expression profiles of total RNA from these fractions, we found that MV was most enriched in microbiome RNA (76.2% of total reads on average), whereas EV-D was notably enriched in human RNA (70.3% of total reads on average). As for human RNA composition, CFS and EV-D were both enriched in snoRNA and tRNA compared with the two EV fractions (EXO and MV, P < 0.05). Interestingly, EXO and MV had highly correlated expression profiles for various noncoding RNAs such as miRNA, tRNA, and yRNA. Our study revealed unique characteristics of circulating RNAs in various saliva fractions, which provides a guideline on preparing saliva samples to study specific RNA biomarkers of interest. [ABSTRACT FROM AUTHOR]

Objective: We aimed to explore the role of long intergenic non‐protein coding RNA 460 (LINC00460) in tongue squamous cell carcinoma (TSCC). Methods: We enrolled 27 TSCC patients to explore LINC00460 expression in clinical TSCC samples. RT‐qPCR measured expression of molecules in this research. Loss‐of‐function assays explored biological function of LINC00460 in TSCC cells. RNA pull‐down assay, luciferase reporter assay, and RIP assay investigated mechanism of LINC00460 underlying TSCC cells. Results: TSCC tissues and cell lines both showed high expression of LINC00460. Functionally, LINC00460 downregulation inhibited TSCC cell growth and promoted TSCC cell apoptosis. Additionally, LINC00460 silencing suppressed tumor growth in vivo. Mechanistically, LINC00460 bound with microRNA 320b (miR‐320b) in TSCC cells. MiR‐320b overexpression suppressed TSCC cell growth and promoted TSCC cell apoptosis. Moreover miR‐320b targeted insulin‐like growth factor 2 mRNA‐binding protein 3 (IGF2BP3) 3′untranslated region in TSCC cells. Furthermore, IGF2BP3 silencing suppressed TSCC cell growth and promoted TSCC cell apoptosis. IGF2BP3 upregulation countervailed effects of silenced LINC00460 on TSCC cells. The LINC00460/miR‐320b/IGF2BP3 axis was associated with lymph node metastasis of TSCC patients. Conclusion: Our research illustrated that LINC00460 facilitated TSCC progression via the miR‐320b/IGF2BP3 axis, highlighting a potential insight for the treatment of TSCC. [ABSTRACT FROM AUTHOR]

Background: Dimethyl fumarate (DMF) is an effective drug for multiple sclerosis and can improve the cognitive dysfunction caused by streptozotocin, but the effect on cognitive dysfunction caused by hypothyroidism is unclear. Methods: After the hypothyroidism rat model induced by propylthiouracil, we gave rats 25 mg/kg DMF by gavage. The body weight during model building and administration was recorded. The levels of T4 and T3 in serum were detected by an automatic biochemical analyzer. Morris water maze test was used to detect the effect of DMF on cognitive learning ability. The effect of DMF on Nissl bodies in the brain tissue was evaluated by Nissl staining. The mRNA and protein levels of BDNF in brain tissue were detected by quantitative reverse transcription-polymerase chain reaction and Western blot. The degrees of p-AKT/AKT and p-CREB/CREB in brain tissue were detected by Western blot. Results: After DMF treatment, the body weight of hypothyroid rats recovered, and the levels of T3 and T4 in the serum were ameliorated. DMF also reduced the escape latency and distance traveled, and increased the swim speed. The number of Nissl bodies and expression of BDNF, p-AKT/AKT, and p-CREB/CREB in the brain tissue were increased after DMF treatment. Conclusion: DMF improved the cognitive dysfunction of hypothyroid rats by increasing the level of BDNF in the brain tissue of hypothyroid rats. [ABSTRACT FROM AUTHOR]

A previous study has elucidated that circular RNA circCLK3 acts as an oncogenic gene in cervical cancer. However, the role and regulatory mechanism of circCLK3 in tongue squamous cell carcinoma (TSCC) remain unknown. Quantitative real‐time PCR was used to examine targeted gene expression in different groups. Cell viability and proliferation were investigated by MTT and 5‐ethynyl‐2′‐deoxyuridine assays. Cell migration and invasion were detected by Transwell assays, and cell apoptosis was measured by flow cytometry analysis. The interaction among genes was investigated using luciferase reporter assay, RNA pull‐down assay, and RNA immunoprecipitation assay. In the present study, our findings revealed the upregulated expression of circCLK3 in TSCC tissues and cell lines. CircCLK3 knockdown suppressed cell proliferation, migration invasion, and induced cell cycle arrest at G0/G1 phase in TSCC. Moreover, circCLK3 acted as a molecular sponge for miR‐455‐5p. PARVA was the target gene of miR‐455‐5p. Furthermore, the negative correlation between expression of miR‐455‐5p and circCLK3 or PARVA in TSCC tissues was discovered. Rescue assays indicated that PARVA overexpression reversed the circCLK3 knockdown‐mediated inhibitory effects on the progression of TSCC. In summary, circCLK3 exerts its carcinogenic effects on TSCC progression via absorbing miR‐455‐5p to upregulate PARVA, which expands our knowledge on the underlying mechanism of TSCC. [ABSTRACT FROM AUTHOR]

Introduction: KLRB1 is a gene encoding CD161 expressed in NK cells and some T cell subsets. At present, KLRB1 is believed to affect tumorigenesis and development by regulating the cytotoxicity of NK cells in several cancers. However, there is a lack of systematic reviews of KLRB1 in a variety of malignancies.Objectives: Hence, our research is aimed at providing a relatively comprehensive understanding of the role of KLRB1 in different types of cancer, paving the way for further research on the molecular mechanism and immunotherapy potential of KLRB1.Methods: In this study, we used relevant public databases, including TCGA (The Cancer Genome Atlas), GEO (Gene Expression Omnibus), CCLE (Cancer Cell Line Encyclopedia), GTEx (Genotype Tissue-Expression), and HPA (Human Protein Atlas), to perform a pan-cancer analysis of KLRB1 across 33 types of cancer. We explored the potential molecular mechanism of KLRB1 in clinical prognosis and tumor immunity from the aspects of gene expression, survival status, clinical phenotype, immune infiltration, immunotherapy response, and chemotherapeutic drug sensitivity.Results: KLRB1 was downregulated in 13 cancers while upregulated in kidney cancer. Patients with high expression of KLRB1 have a better prognosis in most types of cancer. Moreover, the KLRB1 expression level is related to TMB and MSI and related to various immune signatures of tumor. The expression of KLRB1 can affect tumor immune cell infiltration. KLRB1 expression level can also affect the sensitivity of chemotherapy drugs.Conclusions: KLRB1 may be a prognostic and immunological biomarker across tumors. At the same time, KLRB1 expression can reflect the sensitivity of cancer patients to chemotherapy drugs. KLRB1 may become a new target for immunotherapy. [ABSTRACT FROM AUTHOR]

More than one‐third of patients with locally advanced cervical cancer do not respond to chemoradiation therapy (CRT). We aimed to characterize the transcriptional landscape of paired human cervical tumors before and during CRT in order to gain insight into the evolution of treatment response and to elucidate mechanisms of treatment resistance. We prospectively collected cervical tumor biopsies from 115 patients both before and 3 weeks into CRT. RNA‐sequencing, Gene Set Enrichment Analysis and HPV gene expression were performed on 20 paired samples that had adequate neoplastic tissue mid‐treatment. Tumors from patients with no evidence of disease (NED) at last follow‐up had enrichment in pathways related to the immune response both pretreatment and mid‐treatment, while tumors from patients dead of disease (DOD) demonstrated enrichment in biosynthetic and mitotic pathways but not in immune‐related pathways. Patients DOD had decreased expression of T‐cell and cytolytic genes and increased expression of PD‐L2 mid‐treatment compared to patients NED. Histological and immunohistochemical analysis revealed a decrease in tumor‐associated lymphocytes (TAL) during CRT in all patients but tumors from patients DOD had a significantly more pronounced decrease in TALs and CD8+ cells mid‐treatment, which was validated in a larger mid‐treatment cohort. Finally, patients DOD retained more HPV E6/E7 gene expression during CRT and this was associated with increased expression of genes driving mitosis, which was corroborated in vitro. Our results suggest that decreased local immune response and retained HPV gene expression may be acting together to promote treatment resistance during CRT in patients with cervical cancer. What's new? Approximately 30% of cervical cancer patients are resistant to chemoradiation therapy but the causes of treatment resistance are unknown. Here the authors profiled gene expression of paired human cervical tumors before and during chemoradiation. Patients who were resistant to treatment and died of the disease showed a decreased local immune response and increased expression of biosynthetic and mitotic pathways. The results point to a combined role of diminished antitumor immunity and maintained expression of human papilloma virus E6/E7 oncogenes in patients failing chemoradiation treatment. [ABSTRACT FROM AUTHOR]

Background: Polygonum cuspidatum of the Polygonaceae family is a traditional medicinal plant with many bioactive compounds that play important roles in human health and stress responses. Research has attempted to identify biosynthesis genes and metabolic pathways in this species, and quantitative real-time PCR (RT-qPCR) has commonly been used to detect gene expression because of its speed, sensitivity, and specificity. However, no P. cuspidatum reference genes have been identified, which hinders gene expression studies. Here, we aimed to identify suitable reference genes for accurate and reliable normalization of P. cuspidatum RT-qPCR data. Results: Twelve candidate reference genes, including nine common (ACT, TUA, TUB, GAPDH, EF-1γ, UBQ, UBC, 60SrRNA, and eIF6A) and three novel (SKD1, YLS8, and NDUFA13), were analyzed in different tissues (root, stem, and leaf) without treatment and in leaves under abiotic stresses (salt, ultraviolet [UV], cold, heat, and drought) and hormone stimuli (abscisic acid [ABA], ethylene [ETH], gibberellin [GA3], methyl jasmonate [MeJA], and salicylic acid [SA]). Expression stability in 65 samples was calculated using the △CT method, geNorm, NormFinder, BestKeeper, and RefFinder. Two reference genes (NDUFA13 and EF-1γ) were sufficient to normalize gene expression across all sample sets. They were also the two most stable genes for abiotic stresses and different tissues, whereas NDUFA13 and SKD1 were the top two choices for hormone stimuli. Considering individual experimental sets, GAPDH was the top-ranked gene under ABA, ETH, and GA3 treatments, while 60SrRNA showed good stability under MeJA and cold treatments. ACT, UBC, and TUB were suitable genes for drought, UV, and ABA treatments, respectively. TUA was not suitable because of its considerable variation in expression under different conditions. The expression patterns of PcPAL, PcSTS, and PcMYB4 under UV and SA treatments and in different tissues normalized by stable and unstable reference genes demonstrated the suitability of the optimal reference genes. Conclusions: We propose NDUFA13 and EF-1γ as reference genes to normalize P. cuspidatum expression data. To our knowledge, this is the first systematic study of reference genes in P. cuspidatum which could help advance molecular biology research in P. cuspidatum and allied species. [ABSTRACT FROM AUTHOR]

Identification of novel prognostic biomarkers typically requires a large dataset which provides sufficient statistical power for discovery research. To this end, we took advantage of the high-throughput data from The Cancer Genome Atlas ( TCGA) to identify a set of prognostic biomarkers in head and neck squamous cell carcinomas ( HNSCC) including oropharyngeal squamous cell carcinoma ( OPSCC) and other subtypes. In this study, we analyzed mi RNA-seq data obtained from TCGA patients to identify prognostic biomarkers for OPSCC. The identified mi RNAs were further tested with an independent cohort. mi RNA-seq data from TCGA was also analyzed to identify prognostic mi RNAs in oral cavity squamous cell carcinoma ( OSCC) and laryngeal squamous cell carcinoma ( LSCC). Our study identified that miR-193b-3p and miR-455-5p were positively associated with survival, and miR-92a-3p and miR-497-5p were negatively associated with survival in OPSCC. A combined expression signature of these four mi RNAs was prognostic of overall survival in OPSCC, and more importantly, this signature was validated in an independent OPSCC cohort. Furthermore, we identified four mi RNAs each in OSCC and LSCC that were prognostic of survival, and combined signatures were specific for subtypes of HNSCC. A robust 4-mi RNA prognostic signature in OPSCC, as well as prognostic signatures in other subtypes of HNSCC, was developed using sequencing data from TCGA as the primary source. This demonstrates the power of using TCGA as a potential resource to develop prognostic tools for improving individualized patient care. [ABSTRACT FROM AUTHOR]

Background: MicroRNAs are small noncoding RNAs about 22 nt long that play key roles in almost all biological processes and diseases. The fluorescent labeling and lipofection are two common methods for changing the levels and locating the position of cellular miRNAs. Despite many studies about the mechanism of DNA/RNA lipofection, little is known about the characteristics, mechanisms and specificity of lipofection of fluorescent-labeled miRNAs. Methods and Results: Therefore, miRNAs labeled with different fluorescent dyes were transfected into adherent and suspension cells using lipofection reagent. Then, the non-specific binding and its mechanism were investigated by flow cytometer and laser confocal microscopy. The results showed that miRNAs labeled with Cy5 (cyanine fluorescent dye) could firmly bind to the surface of adherent cells (Hela) and suspended cells (K562) even without lipofection reagent. The binding of miRNAs labeled with FAM (carboxyl fluorescein) to K562 cells was obvious, but it was not significant in Hela cells. After lipofectamine reagent was added, most of the fluorescently labeled miRNAs binding to the surface of Hela cells were transfected into intra-cell because of the high transfection efficiency, however, most of them were still binding to the surface of K562 cells. Moreover, the high-salt buffer which could destroy the electrostatic interactions did not affect the above-mentioned non-specific binding, but the organic solvent which could destroy the hydrophobic interactions eliminated it. Conclusions: These results implied that the fluorescent-labeled miRNAs could non-specifically bind to the cell surface by hydrophobic interaction. It would lead to significant errors in the estimation of transfection efficiency only according to the cellular fluorescence intensity. Therefore, other methods to evaluate the transfection efficiency and more appropriate fluorescent dyes should be used according to the cell types for the accuracy of results. [ABSTRACT FROM AUTHOR]

Motivation: MicroRNAs (miRNAs) are small non-coding RNAs that are extensively involved in gene expression regulation. One major roadblock in functional miRNA studies is the reliable prediction of genes targeted by miRNAs, as rules defining miRNA target recognition have not been well-established to date. Availability of high-throughput experimental data from a recent CLASH (cross linking, ligation and sequencing of hybrids) study has presented an unprecedented opportunity to characterize miRNA target recognition patterns, which may provide guidance for improved miRNA target prediction.Results: The CLASH data were analysed to identify distinctive sequence features that characterize canonical and non-canonical miRNA target types. Most miRNA targets were of non-canonical type, i.e. without involving perfect pairing to canonical miRNA seed region. Different miRNAs have distinct targeting patterns, and this miRNA-to-miRNA variability was associated with seed sequence composition. Specifically, seed-based canonical target recognition was dependent on the GC content of the miRNA seed. For miRNAs with low GC content of the seed region, non-canonical targeting was the dominant mechanism for target recognition. In contrast to canonical targeting, non-canonical targeting did not lead to significant target downregulation at either the RNA or protein level.Contact: xwang@radonc.wustl.edu [ABSTRACT FROM AUTHOR]

BACKGROUND: Oropharyngeal squamous cell carcinoma (SCC) rates have been increasing significantly in recent years, despite a decreasing incidence of head and neck cancer in general. Oropharyngeal SCC has many characteristics that are distinct from other head and neck cancers, and thus it is important to focus specifically on cancers arising in this region, with the goal of improving patient outcomes. One important goal is to identify those patients who are likely to fail standard therapy and who could potentially benefit from alternative or targeted treatments. METHODS: In the current study, the prognostic value of microRNAs (miRNAs) was evaluated in patients with oropharyngeal SCC. miRNAs are small, noncoding RNAs that are master regulators of many important biological processes. In total, 150 oropharyngeal tumors were analyzed using the recently developed quantitative polymerase chain reaction-based method for miRNA expression profiling. In addition, the expression of miRNAs was also compared with human papillomavirus (HPV) transcriptional activities. RESULTS: The current study identified 6 miRNAs that were found to be significantly associated with cancer survival. A combined expression signature of these miRNAs was prognostic of oropharyngeal SCC, independent of common clinical features or HPV status. CONCLUSIONS: This new miRNA signature was experimentally validated in an independent oropharyngeal SCC cohort. Furthermore, 5 HPV-related miRNAs were identified, which may help to characterize HPV-induced cancers including both oropharyngeal and cervical SCC. Cancer 2013. © 2012 American Cancer Society. [ABSTRACT FROM AUTHOR]

Simple Summary: Triple-negative breast cancer (TNBC) is the most refractory subtype of breast cancer. Immune checkpoint inhibitor (ICI) therapy has made progress in TNBC treatment. PD-L1 expression is a useful biomarker of ICI therapy efficacy. However, tumor-immune microenvironment (TIME) factors, such as immune cell compositions and tumor-infiltrating lymphocyte (TIL) status, also influence tumor immunity. Therefore, it is necessary to seek biomarkers that are associated with multiple aspects of TIME in TNBC. In this study, we developed an immune-related gene prognostic index (IRGPI) with a substantial prognostic value for TNBC. Moreover, the results from multiple cohorts reproducibly demonstrate that IRGPI is significantly associated with immune cell compositions, the exclusion and dysfunction of TILs, as well as PD-1 and PD-L1 expression in TIME. Therefore, IRGPI is a promising biomarker closely related to patient survival and TIME of TNBC and may have a potential effect on the immunotherapy strategy of TNBC. Tumor-immune cell compositions and immune checkpoints comprehensively affect TNBC outcomes. With the significantly improved survival rate of TNBC patients treated with ICI therapies, a biomarker integrating multiple aspects of TIME may have prognostic value for improving the efficacy of ICI therapy. Immune-related hub genes were identified with weighted gene co-expression network analysis and differential gene expression assay using The Cancer Genome Atlas TNBC data set (n = 115). IRGPI was constructed with Cox regression analysis. Immune cell compositions and TIL status were analyzed with CIBERSORT and TIDE. The discovery was validated with the Molecular Taxonomy of Breast Cancer International Consortium data set (n = 196) and a patient cohort from our hospital. Tumor expression or serum concentrations of CCL5, CCL25, or PD-L1 were determined with immunohistochemistry or ELISA. The constructed IRGPI was composed of CCL5 and CCL25 genes and was negatively associated with the patient's survival. IRGPI also predicts the compositions of M0 and M2 macrophages, memory B cells, CD8+ T cells, activated memory CD4 T cells, and the exclusion and dysfunction of TILs, as well as PD-1 and PD-L1 expression of TNBC. IRGPI is a promising biomarker for predicting the prognosis and multiple immune characteristics of TNBC. [ABSTRACT FROM AUTHOR]

The evaluation of specific endogenous transcript levels is important for understanding transcriptional regulation. More specifically, it is useful for independent confirmation of results obtained by the use of microarray analysis or RNA-seq and for evaluating RNA interference (RNAi)-mediated gene knockdown. Designing specific and effective primers for high-quality, moderate-throughput evaluation of transcript levels, i.e., quantitative, real-time PCR (qPCR), is nontrivial. To meet community needs, predefined qPCR primer pairs for mammalian genes have been designed and sequences made available, e.g., via PrimerBank. In this work, we adapted and refined the algorithms used for the mammalian PrimerBank to design 45,417 primer pairs for 13,860 Drosophila melanogaster genes, with three or more primer pairs per gene. We experimentally validated primer pairs for ~300 randomly selected genes expressed in early Drosophila embryos, using SYBR Green-based qPCR and sequence analysis of products derived from conventional PCR. All relevant information, including primer sequences, isoform specificity, spatial transcript targeting, and any available validation results and/or user feedback, is available from an online database (www.flyrnai.org/flyprimerbank). At FlyPrimerBank, researchers can retrieve primer information for fly genes either one gene at a time or in batch mode. Importantly, we included the overlap of each predicted amplified sequence with RNAi reagents from several public resources, making it possible for researchers to choose primers suitable for knockdown evaluation of RNAi reagents (i.e., to avoid amplification of the RNAi reagent itself). We demonstrate the utility of this resource for validation of RNAi reagents in vivo.

Background: Glycolysis is an important metabolic oncogenic change also play a pivot role in the Warburg effect. Glycolysis related gene PKM2 expressed differently individually. Presently, we sought to investigate the effect of single nucleotide polymorphism (SNP) at rs61991156 of miR-379 on gastric cancer (GC) proliferation and metabolism. Methods: The genotype of rs61991156 in miR-379 was investigated by using real-time PCR. The glycolysis-related metabolites were determined by using GC–TOF–MS. The biological effects of rs61991156 in miR-379 was explored by in vitro studies. Results: In this study, we found that rs61991156 in miR-379 was involved in the occurrence of GC by acting on the 3′UTR region of PKM2. The clinical data analysis revealed that A > G in rs187960998 was significantly associated with better differentiation, small tumor size, and non-metastasis. In vitro study further revealed that A > G SNP of miR-379 could decrease GC cell proliferation as well as the promoter activity and expression of PKM2. The glycolysis of the patients with miR-379 GG genotype was significantly lower than AG and AA genotype by metabolomics analysis. The patients with AA genotype have significantly lower PKM2 expression compared to the G carrier, while there is no significant expression difference in miR-379 expression. Patients with AA genotype have significantly shorter survival rate compared to the G carrier. Conclusion: rs61991156 in miR-379 was highly associated with a decreased risk, well differentiation and better post-surgery survival in Chinese population by inhibiting the expression of PKM2. [ABSTRACT FROM AUTHOR]

Recently we found that ATP5J was over-expressed in tissue samples from patients with colorectal cancer. However, the clinical significance and function of the over-expression of ATP5J in these patients remains unclear. We investigated these issues in the current study. Our results indicated that expression of ATP5J was significantly higher in colorectal cancer tissue than in adjacent tissue, and it was also significantly higher in metastatic lymph nodes than in primary cancer tissue (P<0.05). A correlation between ATP5J expression and tumor differentiation was detected, but no correlation with gender, age, T stage, lymph node metastasis, or survival status was observed. Down-regulation of ATP5J expression attenuated the ability of cell migration and increased the sensitivity to 5-fluorouracil (5-Fu) in cells of the DLD1 cell line. Inversely, up-regulation of ATP5J expression enhanced cell migration and decreased 5-Fu sensitivity, suggesting that the function of ATP5J in colorectal cancer might involve cell migration and 5-Fu sensitivity. [ABSTRACT FROM AUTHOR]

Xylogen-like arabinogalactan protein (XYLP) is an atypical lipid transport protein. In this study, 23 Phyllostachys edulis XYLPs were identified, and their proteins contain characteristic structures of AGP and nsLTP domain. All PeXYLPs can be divided into four clades, and their genes were unevenly distributed on 11 chromosome scaffolds. Collinear analysis revealed that segmental duplication was the main driver for PeXYLP family expansion. The c is-acting elements presented in the promoter are involved in various regulations of PeXYLPs expression. G.O. annotation revealed that PeXYLPs are mainly interested in lipid transport and synthesis and primarily function at the plasma membrane. Transcriptome analysis revealed that PeXYLPs were spatiotemporally expressed and displayed significant variability during various tissue development. Besides that, some PeXYLP s also respond to multiple phytohormones and abiotic stresses. By semi-quantitative RT-PCR, the response of some PeXYLPs to MeJA was confirmed, and the proteins were shown to localize to the plasma membrane mainly. WGCNA in defined regions of fast-growing bamboo shoots revealed that 5 PeXYLPs in 4 gene co-expression modules showed a positive module-trait relationship with three fast-growing regions. This systematic analysis of the PeXYLP family will provide a foundation for further insight into the functions of individual PeXYLP in a specific tissue or organ development, phytohormone perception, and stress responses in the future. • Twenty-three XYLP genes (PeXYLPs) were identified in the genome of Phyllostachys edulis and were classified into four evolutionary clades. • Segmental duplication plays a dominant role in expanding the PeXYLP family in Phyllostachys edulis. • Large-scale expansion of the XYLP family could have occurred after the segregation of monocotyledons and dicotyledons and then expanded independently. • Expression of some PeXYLP genes showed positive correlations with fast growth and elongation of internode in Phyllostachys edulis. [ABSTRACT FROM AUTHOR]

AbstractCentral nervous system (CNS) axons recover poorly following injury because of the expression of myelin-derived inhibitors of axonal outgrowth such as Nogo, myelin-associated glycoprotein (MAG), and oligodendrocyte-myelin glycoprotein (OMgp), all of which bind to the Nogo-66 receptor 1 (NgR1). Herein we examine the role of NgR1 in the recovery of motor and cognitive function after traumatic brain injury (TBI) using a controlled cortical impact (CCI) model in NgR1 knockout (KO) and wild-type (WT) mice. Four weeks post-injury, scores on the Novel Object Recognition test were significantly increased in NgR1 KO mice compared with WT mice (p<0.05), but motor behavior test scores did not differ significantly between the two groups. Nissl staining showed that NgR1 KO mice had less brain injury volume 2 weeks after CCI (p<0.05). Histological analysis revealed more doublecortin (DCX+) cells (p<0.01) and more Ki-67+ cells in the contralateral dentate gyrus (DG) (p<0.05) 2 weeks after CCI in NgR1 KO mice than in WT. Furthermore, DCX+ cells still retained their longer processes in KO mice (p<0.01) 4 weeks following trauma. The number of bromodeoxyuridine (BrdU)+ cells did not differ between the two groups at 4 weeks post-trauma, but KO mice had higher numbers of cells that co-stained with NeuN, a marker of mature neurons. Increased transcription of growth-associated protein (GAP)-43 in both the injured and contralateral sides of the hippocampus (both p<0.05) was detected in NgR1 KO mice relative to WT. These data suggest that NgR1 negatively influences plasticity and cognitive recovery after TBI. [ABSTRACT FROM AUTHOR]

Abstract: Lipin family proteins (lipin 1, 2, and 3) are bifunctional intracellular proteins that regulate metabolism by acting as coregulators of DNA-bound transcription factors and also dephosphorylate phosphatidate to form diacylglycerol [phosphatidate phosphohydrolase activity] in the triglyceride synthesis pathway. Herein, we report that lipin 1 is enriched in heart and that hearts of mice lacking lipin 1 (fld mice) exhibit accumulation of phosphatidate. We also demonstrate that the expression of the gene encoding lipin 1 (Lpin1) is under the control of the estrogen-related receptors (ERRs) and their coactivator the peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α). PGC-1α, ERRα, or ERRγ overexpression increased Lpin1 transcription in cultured ventricular myocytes and the ERRs were associated with response elements in the first intron of the Lpin1 gene. Concomitant RNAi-mediated knockdown of ERRα and ERRγ abrogated the induction of lipin 1 expression by PGC-1α overexpression. Consistent with these data, 3-fold overexpression of PGC-1α in intact myocardium of transgenic mice increased cardiac lipin 1 and ERRα/γ expression. Similarly, injection of the β2-adrenergic agonist clenbuterol induced PGC-1α and lipin 1 expression, and the induction in lipin 1 after clenbuterol occurred in a PGC-1α-dependent manner. In contrast, expression of PGC-1α, ERRα, ERRγ, and lipin 1 was down-regulated in failing heart. Cardiac phosphatidic acid phosphohydrolase activity was also diminished, while cardiac phosphatidate content was increased, in failing heart. Collectively, these data suggest that lipin 1 is the principal lipin protein in the myocardium and is regulated in response to physiologic and pathologic stimuli that impact cardiac metabolism. [Copyright &y& Elsevier]

• Based on our research, reducing LCN2 could effectively induce ferroptosis and hinder the growth of NSCLC. • We explored the effects of LCN2 on NSCLC function and suggested that it may have potential therapeutic effects on NSCLC. • At the cell biological level, we demonstrated that interfering with LCN2 inhibited NSCLC proliferation and migration by promoting ferroptosis, suggesting that it may be a new potential target pathway. Tumor progression is intricately linked to ferroptosis, a recently discovered form of regulated cell death. However, the specific causes of ferroptosis in non-small cell lung cancer (NSCLC) remain unclear. In this study, we conducted transcriptome sequencing on NSCLC samples and identified Lipocalin-2 (LCN2) as a significantly differentially expressed gene associated with ferroptosis in NSCLC. Through the intersection of the set of significantly different genes with ferroptosis-related genes, we unveiled the relevance of LCN2 in NSCLC. To validate our findings, several cell lines (BEAS-2B, A549, H1299, PC-9, H1975) were utilized, and Western blot (WB) analysis was performed. We employed a variety of assays, including CCK8, EDU, scratch, Transwell, and specific assays targeting ferroptosis, to investigate the effects of LCN2 on NSCLC cell proliferation, migration, and ferroptosis. Additionally, LCN2 was evaluated in vivo using a mouse tumor xenograft model. In both NSCLC patients and cells, LCN2 exhibited upregulation and was associated with a poor prognosis. Inhibition of LCN2 promoted ferroptosis, resulting in the inhibition of NSCLC proliferation and migration. Conversely, the ferroptosis inhibitor Fer-1 promoted NSCLC cell proliferation and migration while inhibiting ferroptosis. Furthermore, down-regulating LCN2 reduced Fer-1′s promotion of NSCLC cell migration and proliferation, as well as its prevention of ferroptosis. In vivo inhibition of LCN2 prevented NSCLC cell growth and enhanced ferroptosis. Based on our research, reducing LCN2 could effectively induce ferroptosis and hinder the growth of NSCLC. [ABSTRACT FROM AUTHOR]

Recent research has highlighted the versatile functions of long non-coding RNAs (lncRNAs) in the onset and progression of various malignancies. Still, insufficient knowledge is available on how lnc-SOX9–4 functions in colorectal cancer (CRC) progression. Bioinformatics analysis was used to identify a novel lncRNA (lnc-SOX9–4), and the expression pattern of the RNA in CRC was verified using qRT-PCR. Gene ontology (GO) term analysis and Gene set enrichment analysis (GSEA) were implemented for the identification of the related mechanisms and roles of lnc-SOX9–4. Immune infiltration analysis was conducted for assessment of how lnc-SOX9–4 is linked to tumor immune cell infiltration level. Both in vitro and in vivo phenotype analyses were conducted for scrutinizing how lnc-SOX9–4 impacts the proliferation and metastasis of CRC. RNA pulldown, mass spectrometry analysis, fluorescent in situ hybridization (FISH), western blotting, and RIP assay aided in verifying lnc-SOX9–4 mechanisms linked to CRC progression. An upregulation of lnc-SOX9–4 was observed in the sample CRC cells and tissues. Elevated lnc-SOX9–4 levels showed a positive association with poor clinical prognosis. Lnc-SOX9–4 was closely correlated to several types of immune infiltrating cells. Functionally, the knockdown of lnc-SOX9–4 significantly inhibited CRC cell proliferation, migration, and invasion abilities. Mechanistically, YBX1 was identified as lnc-SOX9–4, specifically interacting protein in the nucleus. Lnc-SOX9–4 could stabilize YBX1 protein levels by inhibiting poly-ubiquitination and degradation of YBX1. Furthermore, phenotype rescue experiments reveal that lnc-SOX9–4 enhanced the CRC cellular potential to proliferate and metastasize by regulating YBX1 levels. Lnc-SOX9–4 promoted CRC progression by suppressing cytoplasmic translocation and promoting protein levels of YBX1 can serve as novel treatment targets for diagnosing and treating CRC. [ABSTRACT FROM AUTHOR]

Asthma is a common chronic disease mainly occurs from childhood. Increased airway smooth muscle mass is involved in the pathogenesis of asthma. Fibulin-5 was upregulated in the lung tissues of patients with COPD and idiopathic pulmonary fibrosis. This study aimed to investigate Fibulin-5 expression in asthmatic patients and the effect and mechanism of Fibulin-5 on the proliferation and migration of airway smooth muscle cells (ASMCs). The expression of Fibulin-5, YAP, and TAZ in the induced sputum of 38 asthmatic children (19 mild and 19 moderate asthmatics) and 19 healthy controls was determined. The effects and mechanisms of Fibulin-5 on the proliferation and migration of ASMCs were analyzed through upregulating Fibulin-5. We found compared with healthy controls, the expression of Fibulin-5, YAP, and TAZ was increased in the induced sputum of asthmatic children and much higher in moderate asthmatics. Fibulin-5 overexpression promoted the proliferation and migration of ASMCs, upregulated the expression of YAP and TAZ, and reduced the levels of p-YAP and p-TAZ. YAP inhibitor (Peptide 17) abrogated the proliferation and migration of ASMCs induced by Fibulin-5 overexpression in a dose-dependent manner. Additionally, Fibulin-5 overexpression enhanced its binding capacity of β1 integrin, and β1 integrin blocking antibody partly reversed the effect of Fibulin-5 overexpression on the levels of YAP and TAZ. In conclusion, Fibulin-5 expression is correlated with the pathogenesis of childhood asthma. It may function at least partly through binding to β1 integrin and modulating Hippo-YAP/TAZ pathway to promote the proliferation and migration of ASMCs. [ABSTRACT FROM AUTHOR]

Purpose: Patients with human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinoma (OPSCC) have a favorable prognosis. As a result, de-escalation clinical trials are under way. However, approximately 10% of patients will experience distant recurrence even with standard-of-care treatment. Here, we sought to identify novel biomarkers to better risk-stratify HPV-positive patients with OPSCC.Methods and Materials: Gene expression profiling by RNA sequencing (RNA-seq) and quantitative polymerase chain reaction was performed on HPV-positive OPSCC primary tumor specimens from patients with and without distant metastasis (DM).Results: RNA-seq analysis of 39 HPV-positive OPSCC specimens revealed that patients with DM had 2-fold higher E6 gene expression levels than did patients without DM (P=.029). This observation was confirmed in a validation cohort comprising 93 patients with HPV-positive OPSCC. The mean normalized E6 expression level in the 17 recurring primary specimens was 13 ± 2 compared with 8 ± 1 in the remaining 76 nonrecurring primaries (P=.001). Receiver operating characteristic analysis established an E6 expression level of 7.3 as a cutoff for worse recurrence-free survival (RFS). Patients from this cohort with high E6 gene expression (E6-high) (n=51, 55%) had more cancer-related deaths (23% vs 2%, P<.001) and DM (26% vs 5%, P<.001) than did patients with low E6 gene expression (E6-low) (n=42, 45%). Kaplan-Meier survival analysis revealed that E6-high had worse RFS (95% vs 69%, P=.004) and cancer-specific survival (97% vs 79%, P=.007). E6-high maintained statistical significance in multivariate regression models balancing surgery, chemotherapy, nodal stage, and smoking status. Gene set enrichment analysis demonstrated that tumors with high E6 expression were associated with P53, epidermal growth factor receptor, activating transcription factor-2, and transforming growth factor-β signaling pathways.Conclusion: High E6 gene expression level identifies HPV-positive OPSCC patients with 5-fold greater risk of distant disease recurrence and worse cancer-specific survival. Validation in a multi-institutional prospective clinical trial is required to assess the utility of E6 gene expression as a clinically useful prognostic biomarker. [ABSTRACT FROM AUTHOR]

Abstract: Objectives: The aim of this study was to determine whether altered calreticulin expression and distribution contribute to the pathogenesis of atrial fibrillation (AF) associated with valvular heart disease (VHD). Background: AF affects electrophysiological and structural changes that exacerbate AF. Atrial remodeling reportedly underlies AF generation, but the precise mechanism of atrial remodeling in AF remains unclear. Methods: Right and left atrial specimens were obtained from 68 patients undergoing valve replacement surgery. The patients were divided into sinus rhythm (SR; n =25), paroxysmal AF (PaAF; n =11), and persistent AF (PeAF; AF lasting >6months; n =32) groups. Calreticulin, integrin-α5, and transforming growth factor-β1 (TGF-β1) mRNA and protein expression were measured. We also performed immunoprecipitation for calreticulin with either calcineurin B or integrin-α5. Results: Calreticulin, integrin-α5, and TGF-β1 mRNA and protein expression were increased in the AF groups, especially in the left atrium in patients with mitral valve disease. Calreticulin interacted with both calcineurin B and integrin-α5. Integrin-α5 expression correlated with TGF-β1 expression, while calreticulin expression correlated with integrin-α5 and TGF-β1 expression. Despite similar cardiac function classifications, calreticulin expression was greater in the PeAF group than in the SR group. Conclusions: Calreticulin, integrin-α5, and TGF-β1 expression was increased in atrial tissue in patients with AF and was related to AF type, suggesting that calreticulin is involved in the pathogenesis of AF in VHD patients. [Copyright &y& Elsevier]

Highlights: [•] Carbon ion radiation increased the oxidative stress in zebrafish embryos. [•] Carbon ion radiation induced transcriptional level effects. [•] The transcriptional level displayed more sensitivity to low dose radiation than the antioxidant enzyme activities. [•] FA induced radioprotective effects by the inhibition of oxidative stress. [Copyright &y& Elsevier]