Two-fold Decrease In Expression Research Articles

Preliminary study of microRNA expression profiling in non-metastatic colorectal cancer.

e15528 Background: MicroRNAs (miRNAs) are non-coding single-stranded RNAs (18–25 nucleotides in length) that regulate gene expression at the posttranscriptional level by promoting the degradation of mRNAs or by blocking mRNA translation. However, the clinical significance in colon cancer is not yet determined. This study aims to identify differentially expressed miRNAs in colorectal cancer by comparing colon cancerous tissues with normal tissues, exploring the potential roles. Methods: The miRNA expression microarray was employed on tumors from patients diagnosed with adenocarcinoma of the colon. The pilot study included samples from 6 patients (3 women and 3 men), aged 55 to 72 years (mean 62.5±8.4 years). The tumour was located in the rectum, TNM status was as follows: cT (all patients at stage T3), cN (3 patients – N1, 3 patients – Nx), cM (all patients at stage M0). Average tumor size 4.6±2.5 cm. Normal colon tissue was used as a control. The studies were performed with the consent of the patient and according to the protocol accepted by the Bioethics Committee at the Medical University of Lublin No. KE-0254/12/2018. The material was collected during tumor removal surgery, then transported and stored in RNAlater. In the next step, microRNAs were isolated using the RNeasy Micro Kit (Qiagen). The quality and quantity of isolated RNA was assessed with NanoDrop 2000c followed by Bioanalyzer 2100 (Agilent), for the next step samples with RIN above 7. The microRNA expression profile was then evaluated using the GeneChip™ miRNA 4.0 Array (Applied Biosystems). Results: On the basis of a pilot microarray study, the profile of differentially expressed miRNAs was successfully screened. 1.39% of 36 222 tested genes showed a significant at least twofold increase in expression, while 1.75% showed a significant at least twofold decrease in expression. Three novel, not yet described in literature, miRNAs i.e. miR-3154 (p-value 0.015), miR-3921 (p-value 0.036), and miR-5006 (p-value 0.007) appeared to be involved in colorectal carcinogenesis. Conclusions: The present study suggests that mentioned miRNAs after confirmation on a greater group of patients may be used as diagnostic markers for the detection of early stage colon cancer in the general population. This paper was created as part of the project “The Best of the Best! 4. 0.” implemented under the Operational Programme: Knowledge Education Development 2014-2020 and co-financed by the European Social Fund.

Stress-Related Regulation Is Abnormal in the Psoriatic Uninvolved Skin.

Keratinocyte stress-response of the uninvolved psoriatic epidermis is known to be altered compared to healthy cells. Therefore, we aimed to reveal potential mechanisms underlying this alteration. We compared the expression of annotated cell-stress-related proteins between uninvolved psoriatic and healthy skin using the protein array method. Data were analyzed by the Reactome over-representation test. We found that p27/CDKN1B and cytochrome C showed at least a two-fold increase, while cyclooxygenase-2, indolamine-2,3-dioxygenase-1, serum paraoxonase 1, serum paraoxonase 3, serine-46-phosphorylated tumor protein p53, and superoxide-dismutase-2 showed a two-fold decrease in expression in the uninvolved skin. Over-representation analysis suggested the Forkhead-box protein O (FOXO)-mediated transcription as the most significant pathway affected by the differently expressed cell-stress-related proteins (DECSRPs). DECSRPs indicate increased FOXO-mediated transcription of cell-cycle genes and reduced interleukin-signaling in the psoriatic uninvolved skin. Nuclear positivity of the FOXO-signaling-related p27/CDKN1B and FOXO1 are negatively correlated with the disease severity and showed increased expression in the uninvolved epidermis and also in healthy primary keratinocytes, which were grown on cartilage oligomeric matrix protein-coated surfaces. Our results indicate a cell-cycle inhibitory process, as a stress-related compensatory mechanism in the uninvolved epidermis, that could be responsible for blocking keratinocyte hyperproliferation in the psoriatic uninvolved skin, thus maintaining the symptomless skin phenotype.

Rsp activates expression of the Cnt system in Staphylococcus aureus

BackgroundThe Cnt system is crucial for the optimal import of essential metals in metal-limiting conditions and contributes to virulence in S. aureus. In a screen for regulators of efflux pumps in a phage-based ultra-high-density transposon library, we identified Rsp as a candidate regulator of the cntE gene.ResultsA two-fold decrease in expression of all genes of the cnt operon was observed by RT-qPCR in the rsp mutant compared to the parental strain, indicating that Rsp acts as an activator of the cnt operon. To determine whether the Rsp activation depends on iron, we compared mutant and parent cnt expression under varying metal conditions. A 2-fold reduction in cnt gene expression was detected in the rsp mutant in TSB, and a slightly smaller decrease (1.9, 1.7, and 1.5-fold changes for cntK, cmtA, and cntE respectively) was observed after addition of dipyridyl. The greatest decrease was seen with addition of FeSO4 (4.1, 5.3 and 6.3-fold changes for cntK, cmtA and cntE respectively). These findings suggest that Rsp activates the cnt operon in low and high iron conditions. To study the relationship between Rsp and the cnt repressors Fur and Zur, we created single and double mutants. Both fur and zur single mutants had significant increases in cnt gene expression compared to the parental strain, as did the fur rsp double mutant. The zur rsp double mutant also had a significant increase in cntK expression and a trend in increases in cntA and cntE expression just below statistical significance. Thus, the ability of Fur and Zur to repress cnt gene expression are not eliminated by the presence of Rsp. However, there were significantly smaller increases in cnt gene expression in the double mutants compared to single mutants, suggesting that Rsp activation can still occur in the absence of these repressors. To determine if Rsp directly modulates expression of cnt genes, incubation of purified Rsp caused a DNA-specific band shift for the cntK and cntA promoters.ConclusionsRsp activation may act to maintain basal cellular levels of staphylopine to scavenge free metals when needed, in addition to metal dependent regulation by Fur and Zur.

MRNA expression in low grade serous ovarian cancer: Results of a nanoString assay in a diverse population

ObjectiveMutations in the MAP kinase pathway (KRAS, NRAS, BRAF) are common in low grade serous ovarian carcinoma (LGSOC). The effect of these and other mutations on RNA transcription in this disease is poorly understood. Our objective was to describe patterns of somatic mutations and gene transcription in a racially diverse population with LGSOC. MethodsUtilizing an institutional tumor registry, patients with LGSOC were identified and charts were reviewed. RNA was extracted from available tumor tissue. Commercial tumor profiling results were analyzed with PanCancer pathway nanoString mRNA expression data. Along with nanoString n-Solver software, Chi-squared, Fishers Exact, and Cox proportional hazards models were used for statistical analysis, with significance set at p < 0.05. Results39 patients were identified—20% Black, 43% Hispanic, and 36% non-Hispanic White. 18 patients had commercial somatic DNA test results, and 23 had available tumor tissue for RNA extraction and nanoString analysis. The most common somatic alterations identified was KRAS (11 patients, 61%), followed by ERCC1 and TUBB3 (9 each, 50%). KRAS mutations were less common in smokers (14.3% vs 90.9%, p = 0.002). RNA expression analysis demonstrated a greater than two-fold decrease in expression of HRAS in tumors from older patients (p = 0.04), and a greater than two-fold decrease in the expression of HRAS in recurrent tumors (p = 0.007). No significant differences were seen in somatic testing results, RNA expression analysis, or progression free survival between different racial and ethnic cohorts. ConclusionsSomatic deficiencies in ERCC1, TUBB3, and KRAS are common in LGSOC in a population of minority patients. HRAS demonstrates decreased expression in tumors from older patients and recurrent tumors.

Identification of retinal homeobox (rax) gene-dependent genes by a microarray approach: The DNA endoglycosylase neil3 is a major downstream component of the rax genetic pathway.

The retinal homeobox (rx/rax) gene is a transcription factor expressed in the developing eye field that is necessary for normal eye development. rax is necessary for retinal specification and stem cell development. The genetic program of early retinal development, including rax expression, can be induced in naïve ectoderm by activation of insulin-like growth factor (IGF) signaling. We have undertaken a microarray-based approach to identify rax-dependent IGF-induced genes. We identified 21 IGF-induced genes that exhibit at least a two-fold decrease in expression when rax expression is knocked down. Ten of these genes were expressed in the developing eye, eight were expressed in the ciliary marginal zone of the mature tadpole retina, and four could significantly rescue the rax knockdown phenotype. One of these, the nei endonuclease VIII-like 3 (neil3) gene, rescued the rax knockdown phenotype to a remarkable degree. We found that neil3 is necessary for normal retinal lamination and retinal neuron differentiation. We have identified neil3 as a component of the rax genetic pathway necessary for normal retinal progenitor cell development. neil3 is involved in the base excision DNA repair pathway, suggesting that this pathway is essential for normal rax-dependent progenitor cell development in the mature retina. Developmental Dynamics 247:1199-1210, 2018. © 2018 Wiley Periodicals, Inc.

Molecular Bases of Brain Preconditioning.

Preconditioning of the brain induces tolerance to the damaging effects of ischemia and prevents cell death in ischemic penumbra. The development of this phenomenon is mediated by mitochondrial adenosine triphosphate-sensitive potassium () channels and nitric oxide signaling (NO). The aim of this study was to investigate the dynamics of molecular changes in mitochondria after ischemic preconditioning (IP) and the effect of pharmacological preconditioning (PhP) with the -channels opener diazoxide on NO levels after ischemic stroke in rats. Immunofluorescence-histochemistry and laser-confocal microscopy were applied to evaluate the cortical expression of electron transport chain enzymes, mitochondrial -channels, neuronal and inducible NO-synthases, as well as the dynamics of nitrosylation and nitration of proteins in rats during the early and delayed phases of IP. NO cerebral content was studied with electron paramagnetic resonance (EPR) spectroscopy using spin trapping. We found that 24 h after IP in rats, there is a two-fold decrease in expression of mitochondrial -channels (p = 0.012) in nervous tissue, a comparable increase in expression of cytochrome c oxidase (p = 0.008), and a decrease in intensity of protein S-nitrosylation and nitration (p = 0.0004 and p = 0.001, respectively). PhP led to a 56% reduction of free NO concentration 72 h after ischemic stroke simulation (p = 0.002). We attribute this result to the restructuring of tissue energy metabolism, namely the provision of increased catalytic sites to mitochondria and the increased elimination of NO, which prevents a decrease in cell sensitivity to oxygen during subsequent periods of severe ischemia.

LOXL2 Expression in HCT116 Cells under Normoxia is Regulated by ROS and HIF Signaling

HIF1α is well known for its role in induction of metastasis leading to cancer progression.LOXL2 and LOX can induce epithelial‐to‐mesenchymal transition (EMT) of tumor cells and turn them into more aggressive phenotype. Recent findings show that LOX expression can be activated by both hypoxia and reactive oxygen species (ROS) via HIF1 pathway, suggesting complex regulation of LOX in EMT in vivo. To address this, we studied the mechanism of regulation of LOXL2 gene expression in human colon cancer HCT116 cells with impaired mitochrondrial respiration (SCO2‐/‐ mutant).q‐PCR showed that under normoxic conditions (21% O2) expression of LOXL2 gene in SCO2‐/‐ cells, which have increased ROS production, is 8 times higher than in wild type. Culturing of SCO2‐/‐ cells for 1 week with ROS scavenger N‐acetylcysteine (NAC) or inhibitor of HIF‐α signaling FM19G11 under normoxia led to twofold decrease in LOXL2 expression, suggesting direct involvement of ROS/HIF1 axis in LOXL2 gene expression in this cell line. However, we saw no statistical difference in LOXL2 expression in SCO2‐/‐ cells treated with NAC under the hypoxic conditions (3% O2), confirming that ROS levels are decreased in hypoxia and are less involved in regulation of LOXL2. At the same time treatment of SCO2‐/‐ cells with FM19G11 led to twofold decrease in expression of LOXL2, thus demonstrating the active role of HIF pathway. Altogether these data demonstrate that different pathways can be involved in HIF1 dependent expression of LOXL2 under hypoxia and oxidative stress. This mechanism can play important role in vivo in cancer progression mediated by LOXs.Supported by SFI.