Putative Target Sites Research Articles

Sirt2 Regulates Liver Metabolism in a Sex-Specific Manner.

Sirtuin-2 (Sirt2), an NAD+-dependent lysine deacylase enzyme, has previously been implicated as a regulator of glucose metabolism, but the specific mechanisms remain poorly defined. Here, we observed that Sirt2-/- males, but not females, have decreased body fat, moderate hypoglycemia upon fasting, and perturbed glucose handling during exercise compared to wild type controls. Conversion of injected lactate, pyruvate, and glycerol boluses into glucose via gluconeogenesis was impaired, but only in males. Primary Sirt2-/- male hepatocytes exhibited reduced glycolysis and reduced mitochondrial respiration. RNAseq and proteomics were used to interrogate the mechanisms behind this liver phenotype. Loss of Sirt2 did not lead to transcriptional dysregulation, as very few genes were altered in the transcriptome. In keeping with this, there were also negligible changes to protein abundance. Site-specific quantification of the hepatic acetylome, however, showed that 13% of all detected acetylated peptides were significantly increased in Sirt2-/- male liver versus wild type, representing putative Sirt2 target sites. Strikingly, none of these putative target sites were hyperacetylated in Sirt2-/- female liver. The target sites in the male liver were distributed across mitochondria (44%), cytoplasm (32%), nucleus (8%), and other compartments (16%). Despite the high number of putative mitochondrial Sirt2 targets, Sirt2 antigen was not detected in purified wild type liver mitochondria, suggesting that Sirt2's regulation of mitochondrial function occurs from outside the organelle. We conclude that Sirt2 regulates hepatic protein acetylation and metabolism in a sex-specific manner.

MiR-6721-5p as a natural regulator of Meta-VCL is upregulated in the serum of patients with coronary artery disease

BackgroundCoronary artery disease (CAD), the leading cause of mortality globally, arises from atherosclerotic blockage of the coronary arteries. Meta-vinculin (meta-VCL), a large spliced isoform of VCL, co-localizes in muscular adhesive structures and plays significant roles in cardiac physiology and pathophysiology. This study aimed to identify microRNAs (miRNAs) regulating meta-VCL expression and investigate the expression alterations of the miRNAs of interest and meta-VCL as potential biomarkers in the serum of CAD patients. MethodsBioinformatics tools were employed to select miRNAs targeting meta-VCL. Cell-based ectopic expression analysis and a dual-luciferase assay were used to examine the interactions between miRNAs and meta-VCL. An ELISA assessed the concentrations of interleukin-6 (IL-6), IL-10, and tumor necrosis factor-α (TNF-α). MiRNA and meta-VCL expression patterns and biomarker suitability were evaluated in serum samples from CAD and non-CAD individuals using real-time PCR. A cardiac cell-line data set and CAD blood exosome samples were analyzed using bioinformatics and ROC curve analyses, respectively. ResultsmiR-6721-5p directly interacted with the putative target sites at the 3′-UTR of meta-VCL and regulated its expression. IL-10 and TNF-α concentrations, which may act as anti-inflammatory factors, decreased following miR-6721-5p upregulation and meta-VCL downregulation. Bioinformatics and experimental expression analyses confirmed downregulated meta-VCL expression and upregulated miR-6721-5p expression in CAD samples. ROC curve analysis yielded an AUC score of 0.705 (P = 0.018), indicating the potential suitability of miR-6721-5p as a biomarker for CAD. ConclusionsmiR-6721-5p plays a regulatory role in meta-VCL expression and may contribute to CAD development by reducing anti-inflammatory factors. These findings suggest that miR-6721-5p could serve as a novel biomarker in the pathogenesis of CAD.

Reversible Pulsed Electrical Fields as an In Vivo Tool to Study Cardiac Electrophysiology: The Advent of Pulsed Field Mapping.

During electrophysiological mapping of tachycardias, putative target sites are often only truly confirmed to be vital after observing the effect of ablation. This lack of mapping specificity potentiates inadvertent ablation of innocent cardiac tissue not relevant to the arrhythmia. But if myocardial excitability could be transiently suppressed at critical regions, their suitability as targets could be conclusively determined before delivering tissue-destructive ablation lesions. We studied whether reversible pulsed electric fields (PFREV) could transiently suppress electrical conduction, thereby providing a means to dissect tachycardia circuits in vivo. PFREV energy was delivered from a 9-mm lattice-tip catheter to the atria of 12 swine and 9 patients, followed by serial electrogram assessments. The effects on electrical conduction were explored in 5 additional animals by applying PFREV to the atrioventricular node: 17 low-dose (PFREV-LOW) and 10 high-dose (PFREV-HIGH) applications. Finally, in 3 patients manifesting spontaneous tachycardias, PFREV was applied at putative critical sites. In animals, the immediate post-PFREV electrogram amplitudes diminished by 74%, followed by 78% recovery by 5 minutes. Similarly, in patients, a 69.9% amplitude reduction was followed by 84% recovery by 3 minutes. Histology revealed only minimal to no focal, superficial fibrosis. PFREV-LOW at the atrioventricular node resulted in transient PR prolongation and transient AV block in 59% and 6%, while PFREV-HIGH caused transient PR prolongation and transient AV block in 30% and 50%, respectively. The 3 tachycardia patients had atypical atrial flutters (n=2) and atrioventricular nodal reentrant tachycardia. PFREV at putative critical sites reproducibly terminated the tachycardias; ablation rendered the tachycardias noninducible and without recurrence during 1-year follow-up. Reversible electroporation pulses can be applied to myocardial tissue to transiently block electrical conduction. This technique of pulsed field mapping may represent a novel electrophysiological tool to help identify the critical isthmus of tachycardia circuits.

Identification of a Specific Biomarker of Acinetobacter baumannii Global Clone 1 by Machine Learning and PCR Related to Metabolic Fitness of ESKAPE Pathogens.

Since the emergence of high-risk clones worldwide, constant investigations have been undertaken to comprehend the molecular basis that led to their prevalent dissemination in nosocomial settings over time. So far, the complex and multifactorial genetic traits of this type of epidemic clones have allowed only the identification of biomarkers with low specificity. A machine learning algorithm was able to recognize unequivocally a biomarker for early and accurate detection of Acinetobacter baumannii global clone 1 (GC1), one of the most disseminated high-risk clones. A support vector machine model identified the U1 sequence with a length of 367 nucleotides that matched a fragment of the moaCB gene, which encodes the molybdenum cofactor biosynthesis C and B proteins. U1 differentiates specifically between A. baumannii GC1 and non-GC1 strains, becoming a suitable biomarker capable of being translated into clinical settings as a molecular typing method for early diagnosis based on PCR as shown here. Since the metabolic pathways of Mo enzymes have been recognized as putative therapeutic targets for ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens, our findings highlight that machine learning can also be useful in knowledge gaps of high-risk clones and provides noteworthy support to the literature to identify relevant nosocomial biomarkers for other multidrug-resistant high-risk clones. IMPORTANCE A. baumannii GC1 is an important high-risk clone that rapidly develops extreme drug resistance in the nosocomial niche. Furthermore, several strains have been identified worldwide in environmental samples, exacerbating the risk of human interactions. Early diagnosis is mandatory to limit its dissemination and to outline appropriate antibiotic stewardship schedules. A region with a length of 367 bp (U1) within the moaCB gene that is not subjected to lateral genetic transfer or to antibiotic pressures was successfully found by a support vector machine model that predicts A. baumannii GC1 strains. At the same time, research on the group of Mo enzymes proposed this metabolic pathway related to the superbug's metabolism as a potential future drug target site for ESKAPE pathogens due to its central role in bacterial fitness during infection. These findings confirm that machine learning used for the identification of biomarkers of high-risk lineages can also serve to identify putative novel therapeutic target sites.

Association of Polymorphism within the Putative miRNA Target Site in the 3'UTR Region of the DRD2 Gene with Neuroticism in Patients with Substance Use Disorder.

The study aims at looking into associations between the polymorphism rs6276 that occurs in the putative miRNA target site in the 3′UTR region of the DRD2 gene in patients with substance use disorder (SUD) comorbid with a maniacal syndrome (SUD MANIA). In our study, we did not state any essential difference in DRD2 rs6276 genotype frequencies in the studied samples of SUD MANIA, SUD, and control subjects. A significant result was found for the SUD MANIA group vs. SUD vs. controls on the Neuroticism Scale of NEO FFI test, and DRD2 rs6276 (p = 0.0320) accounted for 1.7% of the variance. The G/G homozygous variants were linked with lower results on the neuroticism scale in the SUD MANIA group because G/G alleles may serve a protective role in the expression of neuroticism in patients with SUD MANIA. So far, there have been no data in the literature on the relationship between the miRSNP rs6276 region in the DRD2 gene and neuroticism (personal traits) in patients with a diagnosis of substance use disorder comorbid with the affective, maniacal type disturbances related to SUD. This is the first report on this topic.

Post-Transcriptional Effects of miRNAs on PCSK7 Expression and Function: miR-125a-5p, miR-143-3p, and miR-409-3p as Negative Regulators.

The regulatory mechanism of PCSK7 gene is still unknown, although its encoded protein PC7 is the most ancient and highly conserved of all proprotein convertases and exhibits enzymatic and non-enzymatic functions in liver triglyceride regulation. Bioinformatics algorithms were used to predict regulatory microRNAs (miRNAs) of PCSK7 expression. This led to the identification of four miRNAs, namely miR-125a-5p, miR-143-3p, miR-409-3p, and miR-320a-3p, with potential binding sites on the 3′-untranslated region (3′-UTR) of human PCSK7 mRNA. The expression patterns of these miRNAs and PCSK7 mRNA were assessed in three different cell lines with quantitative polymerase chain reaction (qPCR), which revealed reciprocal expression patterns between the expression levels of the four selected miRNAs and PCSK7. Next, the interactions and effects of these miRNAs on PCSK7 expression levels were investigated via cell-based expression analysis, dual-luciferase assay, and Western blot analysis. The data revealed that PCSK7 mRNA levels decreased in cells transfected with vectors overexpressing miR-125a-5p, miR-143-3p, and miR-409-3p, but not miR-320a-3p. The dual-luciferase assay demonstrated that the above three miRNAs could directly interact with putative target sites in PCSK7 3′-UTR and regulate its expression, whereas miR-320-3p exhibited no interaction. Western blot analysis further revealed that the overexpression of miR-125a-5p in Huh7 cells inhibits the expression and ability of PC7 to cleave human transferrin receptor 1. Our results support a regulatory role of these miRNAs on PCSK7 expression and function and open the way to assess their roles in the regulation of PC7 activity in vivo in the development of hepatic steatosis.

Production and characterization of a broad-spectrum antimicrobial 5-butyl-2-pyridine carboxylic acid from Aspergillus fumigatus nHF-01

The present study aims at the production optimization, purification, and characterization of a potent broad-spectrum antimicrobial compound (AMC) produced by Aspergillus fumigatus nHF-01 (GenBank Ac. No. MN190286). The culture conditions were optimized for a higher amount of AMC. The AMC was solvent extracted and characterized by UV–Vis, FT–IR, ESI–MS, and 1H-NMR spectroscopy. The MIC, MBC and mode of action were determined against a set of Gram-positive and Gram-negative human pathogenic bacteria. Its antibiofilm, synergistic and cytotoxic effects were also tested. The putative target site of action was evaluated through in silico molecular docking study. The stain A. fumigatus nHF-01 produced the maximum AMC (5-butyl-2-pyridine carboxylic acid) in 2% MEB (w/v) and 4% YE (w/v) at pH 6.0 and 20 °C temperature with 100 rpm agitation for ten days. It caused complete lethality of the Gram-positive and Gram-negative human pathogenic bacteria at a 129 µg/mL dose by rupture and entire dissolution of cell integrity. It showed moderate antibiofilm activity and had a synergistic activity with streptomycin and additive effects with ciprofloxacin and vancomycin. It targets a respiratory enzyme, Quinol-Fumarate Reductase (1l0v), with the highest binding affinities. It had cytotoxicity against human lung carcinoma A549 cell line and was stable up to 100 °C. Thus, the study revealed that the strain A. fumigatus nHF-01 produces a potent broad-spectrum AMC 5-butyl-2-pyridine carboxylic acid that could be used against human food and topical pathogenic bacteria. This is the first report of such a compound produced from the A. fumigatus.

Downregulation of Talin-1 is associated with the increased expression of miR-182-5p and miR-9-5p in coronary artery disease.

Evidence indicates that the dysregulation of extracellular matrix (ECM) components can lead to cardiovascular diseases. The Talin-1 (TLN1) gene is a major component of the ECM, and it mediates integrin adhesion to the ECM. In this study, we aimed to determine microRNAs (miRs) that regulate the expression of TLN1 and determine expression alterations in TLN1 and its targeting miRs in coronary artery disease (CAD). Data sets of CAD and normal samples of blood exosomes were downloaded, and TLN1 was chosen as one of the genes with differential expressions in an in silico analysis. Next, miR-182-5p and miR-9-5p, which have a binding site on 3´-UTR of TLN1, were selected using bioinformatics tools. Then, the miR target site was cloned in the psiCHECK-2 vector, and direct interaction between the miR target site and the TLN1 3'-UTR putative target site was investigated by luciferase assay. The expression of miR-182-5p, miR-9-5p, and TLN1 in the serum samples of CAD and non-CAD individuals was assessed via a real-time quantitative polymerase chain reaction. Our data revealed that miR-182-5p directly regulated the expression of TLN1. Moreover, miR-182-5p and miR-9-5p were significantly upregulated in the CAD group. Hence, both bioinformatics and experimental analyses determined the downregulated expression of TLN1 in the CAD samples. Our findings demonstrated that miR-182-5p and miR-9-5p could play significant roles in TLN1 regulation and participate in CAD development by targeting TLN1. These findings introduce novel biomarkers with a potential role in CAD pathogenesis.

Contribution of the STAT Family of Transcription Factors to the Expression of the Serotonin 2B (HTR2B) Receptor in Human Uveal Melanoma.

Uveal melanoma (UM) remains the most common intraocular malignancy among diseases affecting the adult eye. The primary tumor disseminates to the liver in half of patients and leads to a 6 to 12-month survival rate, making UM a particularly aggressive type of cancer. Genomic analyses have led to the development of gene-expression profiles that can efficiently predict metastatic progression. Among these genes, that encoding the serotonin receptor 2B (HTR2B) represents the most discriminant from this molecular signature, its aberrant expression being the hallmark of UM metastatic progression. Recent evidence suggests that expression of HTR2B might be regulated through the Janus kinase/Signal Transducer and Activator of Transcription proteins (JAK/STAT) intracellular signalization pathway. However, little is actually known about the molecular mechanisms involved in the abnormally elevated expression of the HTR2B gene in metastatic UM and whether activated STAT proteins participates to this mechanism. In this study, we determined the pattern of STAT family members expressed in both primary tumors and UM cell-lines, and evaluated their contribution to HTR2B gene expression. Examination of the HTR2B promoter sequence revealed the presence of a STAT putative target site (5′-TTC (N)3 GAA3′) located 280 bp upstream of the mRNA start site that is completely identical to the high affinity binding site recognized by these TFs. Gene profiling on microarrays provided evidence that metastatic UM cell lines with high levels of HTR2B also express high levels of STAT proteins whereas low levels of these TFs are observed in non-metastatic UM cells with low levels of HTR2B, suggesting that STAT proteins contribute to HTR2B gene expression in UM cells. All UM cell lines tested were found to express their own pattern of STAT proteins in Western blot analyses. Furthermore, T142 and T143 UM cells responded to interleukins IL-4 and IL-6 by increasing the phosphorylation status of STAT1. Most of all, expression of HTR2B also considerably increased in response to both IL-4 and IL-6 therefore providing evidence that HTR2B gene expression is modulated by STAT proteins in UM cells. The binding of STAT proteins to the −280 HTR2B/STAT site was also demonstrated by electrophoretic mobility shift assay (EMSA) analyses and site-directed mutation of that STAT site also abolished both IL-4 and IL-6 responsiveness in in vitro transfection analyses. The results of this study therefore demonstrate that members from the STAT family of TFs positively contribute to the expression of HTR2B in uveal melanoma.

In silico mapping of 14-3-3ζ and Human TRAF interaction

Abstract Recent advances show that the 14-3-3ζ protein participates in several immune regulations and is a unique regulator of IL-17A signal transduction. The IL-17A signal transduction triggers a 14-3-3ζ-TRAF5 dependent and a 14-3-3ζ-TRAF6 dependent intracellular pathway responsible for producing CXCL-1 and IL-6, respectively. Improved IL-17A signaling blockers are desirable in treating autoimmune diseases such as rheumatoid arthritis and lupus. Due to its unique role, 14-3-3ζ is an attractive target to regulate CXCL-1 and IL-6 levels. The aim of this study is to determine interaction sites between the 14-3-3ζ and TRAF (5 and 6) proteins using bioinformatic tools. We used 6EF5.pdb for 14-3-3ζ, 4GJH.pdb for TRAF5, and ILB6.pdb for TRAF6. Using ZDock, we examined the putative site of the 14-3-3ζ interactions on TRAF (5 and 6) with restrictions and without restrictions. The mapped interacting residues were mutated, and an effect on the interaction with 14-3-3ζ was observed. To further evaluate the interaction quality, we utilized Prodigy to measure the binding energy for several possible structures and narrow down the selected interaction sites further. Results indicate that site 479–485 is the putative target site of the 14-3-3ζ-TRAF5 complex with a ΔG of −10.1 kcal/mol and a Kd of 4.00×10−8. By comparison, residues 483–488 on TRAF6 interact with 14-3-3ζ with a ΔG of −19.1 kcal/mol and a Kd of 9.50×10−15. Our results demonstrate the putative binding sites between the 14-3-3ζ and TRAF (5 and 6) proteins. However, our study has the limitation of using the available but incomplete TRAF structures; therefore, lab studies should be used to confirm these results. Our results provide a rationale to investigate the 14-3-3ζ and TRAF proteins further.

MicroRNA-122 contributes to lipopolysaccharide-induced acute kidney injury via down-regulating the vitamin D receptor in the kidney.

Our previous studies showed that vitamin D receptor (VDR) depletion promotes lipopolysaccharide (LPS)-induced acute kidney injury (AKI) in mice, and renal VDR is down-regulated in AKI, but the mechanism of VDR down-regulation is unclear. Nutritional vitamin D deficiency was induced by feeding mice a vitamin D-deficient (VD-D) diet. Mice were injected intraperitoneally with LPS (20mg/kg) to establish LPS-induced AKI. Levels of VDR and miR-122 were measured both in vivo and in vitro. The associations between VDR and miR-122 were analysed by dual-luciferase reporter assays. Compared with vitamin D-sufficient (VD-S) mice, VD-D mice developed more severe renal injury following LPS challenge. LPS induced a dramatic decrease in VDR expression and marked induction of miR-122 both in vivo and in vitro. Furthermore, miR-122 hairpin inhibitor alleviated LPS-induced VDR down-regulation whereas miR-122 mimic directly suppressed VDR expression in HK-2 cells. In luciferase reporter assays, miR-122 mimic was able to suppress luciferase activity in 293T cells co-transfected with a luciferase reporter that contains a putative miR-122 target site from 3'UTR of the VDR transcript, but not when this site was mutated. Moreover, miR-122 mimic significantly blocked paricalcitol-induced luciferase activity in 293T cells co-transfected with a VDRE-driven luciferase reporter, whereas miR-122 hairpin inhibitor enhanced paricalcitol's activity to suppress PUMA and caspase 3 activation induced by LPS in HK-2 cells. Collectively, these studies provide evidence that miR-122 directly targets VDR in renal tubular cells, which strongly suggest that miR-122 up-regulation in the kidney under LPS challenge contributes to kidney injury by down-regulating VDR expression.

FMR1 expression in human granulosa cells and variable ovarian response: control by epigenetic mechanisms.

In humans, FMR1 (fragile X mental retardation 1) is strongly expressed in granulosa cells (GCs) of the female germline and apparently controls efficiency of folliculogenesis. Major control mechanism(s) of the gene transcription rate seem to be based on the rate of CpG-methylation along the CpG island promoter. Conducting CpG-methylation-specific bisulfite-treated PCR assays and subsequent sequence analyses of both gene alleles, revealed three variably methylated CpG domains (FMR1-VMR (variably methylated region) 1, -2, -3) and one completely unmethylated CpG-region (FMR1-UMR) in this extended FMR1-promoter-region. FMR1-UMR in the core promoter was exclusively present only in female GCs, suggesting expression from both gene alleles, i.e., escaping the female-specific X-inactivation mechanism for the second gene allele. Screening for putative target sites of transcription factors binding with CpG methylation dependence, we identified a target site for the transcriptional activator E2F1 in FMR1-VMR3. Using specific electrophoretic mobility shift assays, we found E2F1 binding efficiency to be dependent on CpG-site methylation in its target sequence. Comparative analysis of these CpGs revealed that CpG 94-methylation in primary GCs of women with normal and reduced efficiency of folliculogenesis statistically significant differences. We therefore conclude that E2F1 binding to FMR1-VMR3 in human GCs is part of an epigenetic mechanism regulating the efficiency of human folliculogenesis. Our data indicate that epigenetic mechanisms may control GC FMR1-expression rates.

Genome-Wide Identification and Characterization of the TCP Gene Family in Cucumber (Cucumis sativus L.) and Their Transcriptional Responses to Different Treatments.

TCP proteins are plant-specific transcription factors widely implicated in leaf morphogenesis and senescence, flowering, lateral branching, hormone crosstalk, and stress responses. However, the relationship between the transcription pattern of TCPs and organ development in cucumber has not been systematically studied. In this study, we performed a genome-wide identification of putative TCP genes and analyzed their chromosomal location, gene structure, conserved motif, and transcript expression. A total of 27 putative TCP genes were identified and characterized in cucumber. All 27 putative CsTCP genes were classified into class I and class II. Class I comprised 12 CsTCPs and Class II contained 15 CsTCPs. The 27 putative CsTCP genes were randomly distributed in five of seven chromosomes in cucumber. Four putative CsTCP genes were found to contain putative miR319 target sites. Quantitative RT-PCR revealed that 27 putative CsTCP genes exhibited different expression patterns in cucumber tissues and floral organ development. Transcript expression and phenotype analysis showed that the putative CsTCP genes responded to temperature and photoperiod and were induced by gibberellin (GA)and ethylene treatment, which suggested that CsTCP genes may regulate the lateral branching by involving in multiple signal pathways. These results lay the foundation for studying the function of cucumber TCP genes in the future.

MiR34a Regulates Neuronal MHC Class I Molecules and Promotes Primary Hippocampal Neuron Dendritic Growth and Branching.

In the immune system, Major Histocompatibility Complex class I (MHC-I) molecules are located on the surface of most nucleated cells in vertebrates where they mediate immune responses. Accumulating evidence indicates that MHC-I molecules are also expressed in the central nervous system (CNS) where they play important roles that are significantly different from their immune functions. Classical MHC-I molecules are temporally and spatially expressed in the developing and adult CNS, where they participate in the synaptic formation, remodeling and plasticity. Therefore, clarifying the regulation of MHC-I expression is necessary to develop an accurate understanding of its function in the CNS. Here, we show that microRNA 34a (miR34a), a brain enriched noncoding RNA, is temporally expressed in developing hippocampal neurons, and its expression is significantly increased after MHC-I protein abundance is decreased in the hippocampus. Computational algorithms identify putative miR34a target sites in the 3′UTR of MHC-I mRNA, and here we demonstrate direct targeting of miR34a to MHC-I mRNA using a dual-luciferase reporter assay system. MiR34a targeting can decrease constitutive MHC-I expression in both Neuro-2a neuroblastoma cells and primary hippocampal neurons. Finally, miR34a mediated reduction of MHC-I results in increased dendritic growth and branching in cultured hippocampal neurons. Taken together, our findings identify miR34a as a novel regulator of MHC-I for shaping neural morphology in developing hippocampal neurons.

MiR-200b suppresses TNF-α-induced AMTN production in human gingival epithelial cells.

Amelotin (AMTN) is an enamel protein that is localized in junctional epithelium (JE) of gingiva and suggested to be involved in the attachment between JE and tooth enamel. MicroRNA is a small non-coding RNA that regulates gene expression at post-transcriptional level by binding to the 3'-untranslated region (3'-UTR) of target mRNAs. In this study, we have analyzed the effects of miR-200b on the expression of AMTN in human gingival epithelial (Ca9-22) cells. Total RNAs and proteins were extracted from Ca9-22 cells transfected with miR-200b expression plasmid or miR-200b inhibitor and stimulated by TNF-α (10ng/ml, 12h). AMTN and inhibitor of kappa-B kinase beta (IKKβ) mRNA and protein levels were measured by qPCR and Western blot. Human AMTN 3'-UTR that contains putative miR-200b target sites were cloned downstream of -353AMTN luciferase (LUC) plasmid. Ca9-22 cells were transfected with -353AMTN 3'-UTR LUC constructs and miR-200b expression plasmid, and LUC activities were measured with or without stimulation by TNF-α. TNF-α-induced AMTN mRNA levels were partially inhibited by miR-200b overexpression and enhanced by miR-200b inhibitor. TNF-α-induced IKKβ mRNA and protein levels were almost completely inhibited by miR-200b. Transcriptional activities of -353AMTN 3'-UTR LUC constructs were induced by TNF-α and partially inhibited by miR-200b. IKKβ inhibitor IMD0354 and NF-κB inhibitor triptolide decreased TNF-α-induced LUC activities. Furthermore, both inhibitors reduced AMTN mRNA levels in the presence or absence of TNF-α. These results suggest that miR-200b suppresses AMTN expression by targeting to AMTN and IKKβ mRNAs in the human gingival epithelial cells.

Using multiple-steps bioinformatic analysis to predict the potential microRNA targets by cardiogenic HoxA11.

In this study, our major aim is to using multiple-steps bioinformatic analysis to predict cardiogenic genes with targeting mRNA profiling for predicting cardiogenic HoxA11 gene. We first analyzed the microarray data with bioinformatic measurement, including combining with panel module 1 (mouse embryonic stem cells), panel module 2 (mouse induced pluripotent stem cells), and panel module 3 (gene list form literature of heart development). A literature-based comparison of the two microarrays and a software-based (Targetscan program, www.targetscan.org) comparative analysis of the two datasets. Furthermore, we select the common central pathways and potential candidate genes involved in the cardiomyocyte-lineaged differentiation and development. Schematic presentation of a putative miR181a target site in Hox-A11 3'UTR. The bioinformatic result showed that potential interacted cardiogenic targets of Tbx5, Tbx20, Mal2c, Nkx2.5, cTNT, Cx43, MHC, and MCK in different treatment groups of pluripotent stem cells by using a literature-based comparison of the two microarrays and a software-based gene-lineage system. Our findings support that mir181a is an up-stream regulating microRNA to target the 3'UTR of HoxA11 mRNA during the process of cardiomyocyte differentiation.

Altered expression of CYP17A1 and CYP19A1 in undescended testes of dogs with unilateral cryptorchidism.

Cryptorchidism is the most common disorder of sex development in dogs and testosterone plays a crucial role in the inguinal phase of the testes descending into the scrotum. The molecular background of impaired testosterone synthesis in the testes of cryptorchid dogs is poorly elucidated. In this study, we analyzed the expression of four genes involved in testicular steroidogenesis (CYP17A1, CYP19A1, HSD3B2 and HSD17B3) in undescended and contralateral scrotal testes from inguinal unilateral cryptorchid dogs (n=13) and from the scrotal gonads of normal males (n=15). We found that transcript level of CYP17A1 was significantly increased in inguinal gonads, while the level of CYP19A1 was decreased. For these two genes, we analyzed the methylation level of single CpG sites in the promoter region localized within putative target sites for testicular transcription factors (NUR77, CREB, CAR and HSF2). A correlation between decreased methylation in the promoter of CYP17A1 and its increased transcript level in undescended gonads was observed, but the change in protein level was not significant. We also resequenced the 5'-flanking region of both genes and two known polymorphic sites, SNP in CYP17A1 and an indel in CYP19A1, were found. However, the distribution of the variants in affected (n=80) and control (n=75) dogs was not associated with cryptorchidism. We tentatively conclude that the altered expression of CYP17A1 and CYP19A1 in undescended testes could be caused by their exposure to increased temperature in the body. Furthermore, we showed that the identified polymorphisms cannot be considered markers associated with a predisposition to cryptorchidism.

MiR-122-5p increases radiosensitivity and aggravates radiation-induced rectal injury through CCAR1

Radiation-induced rectal injury is a major side-effect observed in patients with pelvic malignancies who receive radiotherapy. MicroRNA (miRNA), involved in many cellular biological processes, can be disturbed by ionizing radiation (IR). In this study, we have investigated the function of microRNA-122-5p (miR-122-5p) in radiation-induced rectal injury. MiR-122-5p levels in the serum of rectal cancer patients or in the rectal tissues of C57BL/6 mice before and after IR were detected by quantitative real-time PCR (qRT-PCR). We found that the miR-122-5p levels were significantly up-regulated in patients' serum or in mice rectal tissues after IR. Elevation of miR-122-5p levels sensitized human intestinal epithelial crypt (HIEC) cells to IR both in vitro and in vivo. MiR-122-5p mimic was transfected to HIEC cells and the downstream targets were predicted by bioinformatic analysis. Two putative target sites of miR-122-5p in the 3’UTR of the cell cycle and apoptosis regulator 1 (CCAR1) mRNA were found and verified by luciferase reporter assay. Overexpression of miR-122-5p or silencing CCAR1 combined with IR significantly inhibited cell survival, enhanced radiosensitivity, and increased cell apoptosis compared to that in the negative control group in vitro. In vivo injection of miR-122-5p antagomir after IR significantly alleviated radiation-induced rectal injury in mice. These results suggest that miR-122-5p aggravates radiation-induced rectal injury through targeting CCAR1.

DbMTS: A comprehensive database of putative human microRNA target site SNVs and their functional predictions.

MicroRNAs (miRNA) are short noncoding RNAs that can repress the expression of protein-coding messenger RNAs (mRNAs) by binding to the 3'-untranslated region (UTR) of the target. Genetic mutations such as single nucleotide variants (SNVs) in the 3'-UTR of the mRNAs can disrupt miRNA regulation. In this study, we presented dbMTS, a database for miRNA target site (MTS) SNVs and their functional annotations. This database can help studies easily identify putative SNVs that affect miRNA targeting and facilitate the prioritization of their functional importance. dbMTS is freely available for academic use at http://database.liulab.science/dbMTS as a web service or a downloadable attached database of dbNSFP.

Role of GSK3β and the A‐Type Potassium Channel in the Behavioral Phenotype of a Mouse Model of Parkinson’s Disease

Degeneration of dopaminergic (DA) neurons is a hallmark of Parkinson’s disease (PD). The mechanism involved in the site‐specific dysfunction of DA in the substantia nigra pars compacta (SNpc) but not in the ventral tegmental area (VTA) is not well understood. Glycogen Synthase Kinase 3 beta (GSK3β) activity has been shown to be increased in mouse models of PD and implicated in the human disease. Here we use the rotarod test to evaluate locomotion and balance, the open field test to evaluate anxiety and locomotion and whole cell current studies of DA neurons in brain slices of the midbrain to test the hypothesis that increased GSK3β activity in a mouse model for rapidly progressive PD inhibits A‐Type K+ channel activity specifically in the SNpc compared with the VTA, resulting in motor and non‐motor dysfunction in these mice. Using mice that overexpress A53Tα‐synuclein (A53T) under the pituitary specific PITX3 promoter as an animal model for PD, which developed gait disturbances as early as 7 weeks of age, we determined that A53T mice demonstrated decreased rotarod time compared with controls (134.4±27.0 sec (n=15) vs 287.1±13.0 sec (n=30); p < 0.001). Furthermore, treatment for 3 weeks with a GSK3β inhibitor, TWS‐119, resulted in an increase in rotarod time after 1 week (2.4±0.55 fold (n=5); p = 0.043), which was stable for up to 3 weeks. Furthermore, the open field test demonstrated that A53T mice spent more time in the periphery and traveled less in the center as compared to control mice (78.6±16.3 cm (n=17) vs 177.8±24.0 cm (n=30); p=0.006), and treatment with TWS‐119 led to a decrease in time spent in the periphery and an increase in distance traveled in the center as compared to control, indicating that GSK3β inhibition improved motor and non‐motor functions in this model. Whole‐cell patch‐clamp recordings of DA neurons from brain slices of A53T mice demonstrated a nearly 2‐fold increase in the incidence of spontaneous firing of DA neurons in the SNpc in response to current injection compared to WT (34.8±5.5 pA (n=14) vs 16±5.4 pA (n=6); p=0.04), while firing of DA neurons in slices from TWS‐119 treated A53T mice was not significantly different from WT. There was no difference in firing frequency in DA neurons in the VTA from brain slices from WT and A53T mice. Measurements of A‐Type K+ currents in SNpc of brain slices of A53T mice demonstrated a marked decrease in current density compared to WT (87.4±14.0pA (n=8) vs 164.7±32.4pA (n=19); p<0.001), while A‐Type currents in brain slices from TWS‐119 treated mice were not significantly different from control. Moreover, we identified a putative target site of GSK3β in the Kv4.2 structural subunits of the A‐Type K+ channel. These data support the conclusion that hyperactivity of GSK3β might play a role in the development of a PD behavioral phenotype in the PITX3/A53T mouse model for PD by mediating an increase in excitability of DA neurons via inhibition of the A‐type K channel repolarizing current specifically in DA neurons of the SNpc. This suggests the A‐Type K channel and its subunits as potential therapeutic targets in PD.Support or Funding InformationFunded by The Michael J. Fox Foundation