Protein Levels Of Target Genes Research Articles

Inhibition of arachidonate 15-lipoxygenase reduces the epithelial-mesenchymal transition in eosinophilic chronic rhinosinusitis with nasal polyps.

The epithelial-mesenchymal transition (EMT) is a distinguishing characteristic of chronic rhinosinusitis with nasal polyps (CRSwNP). The underlying mechanism remains largely unknown. Arachidonate 15-lipoxygenase (ALOX15), an enzyme involved in arachidonic acid metabolism, has been reported to cause airway epithelial injury and thus may further promote the EMT. The aim of this study was to evaluate the role of ALOX15 during the EMT process in CRSwNP. A total of 54 samples were obtained, including 10 from healthy control, 16 from non-eosinophilic CRSwNP, and 28 from eosinophilic CRSwNP. Hematoxylin and eosin staining was performed to determine the basement membrane (BM) thickness. The concentration of molecules mediating remodeling was assayed by Luminex. The messenger RNA (mRNA) and protein levels of target genes were measured by quantitative real-time polymerase chain reaction (PCR) and Western blotting. EMT was enhanced in eosinophilic CRSwNP compared with the healthy controls and non-eosinophilic CRSwNP infiltrated with lymphocytes and/or plasma cells. The expression pattern of molecules related to remodeling, including matrix metalloproteinases (MMPs), tissue inhibitor of metalloproteinases (TIMPs), and transforming growth factor β (TGF-β) family members, differed between the subtypes of CRSwNP. The mRNA level of ALOX15 was correlated with the BM thickness and MMP-1 and TGF-β3 expression. The inhibition of ALOX15 by PD146176 could induce claudin-1, claudin-4, claudin-7, zonula occludens (ZO)-1, ZO-2, E-Cadherin, TIMP-1, and TIMP-3 expressions and reduce the levels of MMP-1 and N-Cadherin in epithelial cells acquired from eosinophilic CRSwNP patients. The specific inhibition of ALOX15 could attenuate the EMT, which may provide an alternative method for the treatment of CRSwNP.

Bioengineering of single ncRNA molecule for multi‐targeting against NSCLC

Lung cancer is the second most common cancer among both men and women in the United States. The majority of lung cancer cases are classified as non‐small cell lung cancer (NSCLC). Current NSCLC treatments include resection surgery, radiation, and pharmacotherapy which all provide some benefits and have limitations. As a result, lung cancer caused deaths account for about 27% of all cancer deaths in the US. Therefore, new therapeutic agents are highly demanded for NSCLC. MicroRNAs (miRNA or miR) are a class of genome‐derived, small noncoding RNAs (ncRNA) in cells that govern target gene expression through mRNA degradation or translational inhibition. Some miRNAs such as miR‐34a, miR‐124 and let7c are commonly downregulated in NSCLC tissues/cells and target various oncogenes such as CDK6, STAT3, and Ras. Restoring the expression or function of such dysregulated, tumor suppressive miRNAs represents a new strategy for cancer therapy. Aiming at simultaneously introducing multiple miRNAs in NSCLC cells for multi‐targeting, we employed our recently developed ncRNA bioengineering technology to produce a single ncRNA molecule bearing multiple miRNAs. Our data showed that multi‐targeting ncRNA agents were successfully expressed in E. coli. We were able to purify recombinant ncRNAs to a high degree of homogeneity using anion exchange FPLC methods. We further demonstrated that treatment of human NSCLC cells with bioengineered ncRNAs elevated cellular miR‐34a, miR‐124 and let‐7c levels, reduced protein levels of target genes including CDK6, STAT3 and Ras, and consequently inhibited NSCLC cell proliferation. Therefore, using single ncRNA agent for multi‐targeting may be more effective for the treatment of NSCLC.Support or Funding InformationThis study was supported by grants R01GM133888 and U01CA175315 from NIH.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mammalian-enabled (MENA) protein enhances oncogenic potential and cancer stem cell-like phenotype in hepatocellular carcinoma cells.

Mammalian‐enabled (MENA) protein is an actin‐regulatory protein that influences cell motility and adhesion. It is known to play a role in tumorigenicity of hepatocellular carcinoma (HCC) but the underlying molecular mechanism remains unknown. This study aimed to investigate the oncogenic potential of MENA and its capacity to regulate cancer stem cell (CSC)‐like phenotypes in HCC cells. Real‐time‐PCR and western blot were used to assess mRNA and protein levels of target genes in human HCC tissue specimens and HCC cell lines, respectively. Stable MENA‐overexpressing HCC cells were generated from HCC cell lines. Transwell cell migration and colony formation assays were employed to evaluate tumorigenicity. Ectopic expression of MENA significantly enhanced cell migration and colony‐forming ability in HCC cells. Overexpression of MENA upregulated several hepatic progenitor/stem cell markers in HCC cells. A high MENA protein level was associated with high mRNA levels of MENA, CD133, cytokeratin 19 (CK19), and epithelial cell adhesion molecule (EpCAM) in human HCC tissues. Overexpression of MENA enhanced epithelial‐to‐mesenchymal transition (EMT) markers, extracellular signal‐regulated kinases (ERK) phosphorylation, and the level of β‐catenin in HCC cells. This study demonstrated that overexpression of MENA in HCC cells promoted stem cell markers, EMT markers, and tumorigenicity. These effects may involve, at least partially, the ERK and β‐catenin signaling pathways.

Co-inhibition of Notch and NF-κB Signaling Pathway Decreases Proliferation through Downregulating IκB-α and Hes-1 Expression in Human Ovarian Cancer OVCAR-3 Cells.

Background: Ovarian cancer is one of the most lethal gynecological malignancies and numerous changes in signaling cascades are involved in the initiation and progression of ovarian cancerous cells. Here, we investigated the role of NF-κB and Notch pathways inhibition on human ovarian cancer OVCAR-3 cells proliferation and IκB-α and Hes-1 expression as 2 key genes in these pathways regulation. Methods: The effects of Bay 11-7085 and DAPT, NF-κB and Notch pathways specific inhibitors, on cell proliferation were evaluated using MTT assay. In addition, the cells were transfected by Notch and IKK-β siRNAs. mRNA and protein levels of target genes were measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot after 48 h incubation with inhibitors and siRNAs. Results: Bay 11-7085 and DAPT significantly decreased the cell proliferation OVCAR-3. IκB-α and Hes-1 mRNA levels decreased to 5 or 3% and 6% or 2% after treatment with Bay 11-7085 or DAPT, respectively (p<0.05). We also found that combination treatment exert a more potent effects on the expression of these gene (p<0.05). Moreover, siRNA transfection caused a significant reduction in IκB-α and Hes-1 mRNA levels (p<0.05). In the protein level, OVCAR-3 cell treatment with both chemichal inhibitors and specific siRNA cause a significant decrease in the expression of target genes (p<0.05) Conclusion: Our findings suggest that inhibition of NF-κB and Notch signaling pathways can effectively reduce OVCAR-3 cells proliferation. Therefore, pharmacological targeting of the NF-κB and Notch signaling pathway could be a promising future treatment of ovarian cancer.

Early activation of pro-fibrotic WNT5A in sepsis-induced acute lung injury.

IntroductionThe mechanisms of lung repair and fibrosis in the acute respiratory distress syndrome (ARDS) are poorly known. Since the role of WNT/β-catenin signaling appears to be central to lung healing and fibrosis, we hypothesized that this pathway is activated very early in the lungs after sepsis.MethodsWe tested our hypothesis using a three-step experimental design: (1) in vitro lung cell injury model with human bronchial epithelial BEAS-2B and lung fibroblasts (MRC-5) cells exposed to endotoxin for 18 hours; (2) an animal model of sepsis-induced ARDS induced by cecal ligation and perforation, and (3) lung biopsies from patients who died within the first 24 hours of septic ARDS. We examined changes in protein levels of target genes involved in the Wnt pathway, including WNT5A, non-phospho (Ser33/37/Thr41) β-catenin, matrix metalloproteinase-7 (MMP7), cyclin D1, and vascular endothelial growth factor (VEGF) by Western blotting and immunohistochemistry. Finally, we validated the main gene targets of this pathway in experimental animals and human lungs.ResultsProtein levels of WNT5A, non-phospho (Ser33/37/Thr41) β-catenin, total β-catenin, MMP7, cyclin D1, and VEGF increased after endotoxin stimulation in BEAS-2B and MRC-5 cells. Lungs from septic animals and from septic humans demonstrated acute lung inflammation, collagen deposition, and marked increase of WNT5A and MMP7 protein levels.ConclusionsOur findings suggest that the WNT/β-catenin signaling pathway is activated very early in sepsis-induced ARDS and could play an important role in lung repair and fibrosis. Modulation of this pathway might represent a potential target for treatment for septic and ARDS patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s13054-014-0568-z) contains supplementary material, which is available to authorized users.

Suppression of the epithelial-mesenchymal transition by SHARP1 is linked to the NOTCH1 signaling pathway in metastasis of endometrial cancer.

BackgroundMechanisms governing the metastasis of endometrial cancer (EC) are poorly defined. Recent data support a role for Enhancer-of-split and hairy-related protein 1 (SHARP1), a basic helix-loop-helix transcription repressor, in regulating invasiveness and angiogenesis of several human cancers. However, the role of SHARP1 in metastasis of EC remains unclear.MethodsHuman EC cell lines (Ishikawa and HEC-1B) were used. SHARP1 was upregulated by lentivirus transduction, while intracellular domain of NOTCH1 (ICN) were upregulated by transient transfection with plasmids. Effects of SHARP1 on cell migration and invasion were evaluated by wound healing assay and transwell invasion assay. Experimental metastasis assay were performed in nude mice. Effects of SHAPR1 on protein levels of target genes were detected by western blotting. Furthermore, the association between SHARP1 and the NOTCH1/EMT pathway was further verified in EC tissue specimens by immunohistochemical analysis.ResultsOverexpression of SHARP1 in EC cells inhibited cell migration, invasion, and metastasis. Exogenous SHARP1 overexpression affected the proteins levels of genes involved in EMT process and NOTCH1 signaling pathway. Upregulation of ICN in SHARP1-overexpressing Ishikawa cells induced cell migration and an EMT phenotype. Additionally, immunohistochemical analysis demonstrated that SHARP1 protein levels were lower in metastatic EC than in primary tumors, and statistical analysis revealed correlations between levels of SHARP1 and markers of EMT and NOTCH1 signaling pathway in human EC tissue specimen.ConclusionsThis work supports a role for SHARP1 in suppressing EMT and metastasis in EC by attenuating NOTCH1 signaling. Therefore, SHARP1 may be a novel marker for lymphatic metastasis in EC patients.

Amyloid-beta disrupts calcium and redox homeostasis in brain endothelial cells.

In Alzheimer's disease, the accumulation of amyloid-beta (Aβ) in the brain occurs in the parenchyma and cerebrovasculature. Several evidences support that the neuronal demise is potentiated by vascular alterations in the early stages of the disease, but the mechanisms responsible for the dysfunction of brain endothelial cells that underlie these cerebrovascular changes are unknown. Using rat brain microvascular endothelial cells, we found that short-term treatment with a toxic dose of Aβ1-40 inhibits the Ca(2+) refill and retention ability of the endoplasmic reticulum and enhances the mitochondrial and cytosolic response to adenosine triphosphate (ATP)-stimulated endoplasmic reticulum Ca(2+) release. Upon prolonged Aβ1-40 exposure, Ca(2+) homeostasis was restored concomitantly with a decrease in the levels of proteins involved in its regulation operating at the plasma membrane, endoplasmic reticulum, and mitochondria. Along with perturbations in Ca(2+) regulation, an early increase in the levels of oxidants and a decrease in the ratio between reduced and oxidized glutathione were observed in Aβ1-40-treated endothelial cells. Under these conditions, the nuclear levels of oxidative stress-related transcription factors, namely, hypoxia-inducible factor 1α and nuclear factor (erythroid-derived 2)-related factor 2, were enhanced as well as the protein levels of target genes. In conclusion, Aβ1-40 affects several mechanisms involved in Ca(2+) homeostasis and impairs the redox homeostasis simultaneously with stimulation of protective stress responses in brain endothelial cells. However, the imbalance between cell death and survival pathways leads to endothelial dysfunction that in turn contributes to cerebrovascular impairment in Alzheimer's disease.

MicroRNA-202-3p Inhibits Cell Proliferation by Targeting ADP-Ribosylation Factor-like 5A in Human Colorectal Carcinoma

MicroRNAs (miRNA) that are strongly implicated in carcinogenesis have recently reshaped our understanding of the role of non-protein-coding RNAs. Here, we focused on the function and molecular mechanism of miR-202-3p and its potential clinical application in colorectal cancer. miR-202-3p expression was determined by quantitative reverse transcriptase PCR (qRT-PCR) in 94 colorectal cancer tissues and corresponding noncancerous tissues (NCT). Cell proliferation and colony formation assays in vitro and xenograft experiments in vivo were used to evaluate the effect of miR-202-3p on colorectal cancer cell proliferation. Luciferase assay and Western blot analysis were performed to validate the potential targets of miR-202-3p after the preliminary screening by online prediction and microarray analysis. The mRNA and protein levels of target genes were detected by qRT-PCR and immunohistochemical staining. The copy number of pre-miR-202 was measured by quantitative PCR. First, miR-202-3p was significantly downregulated in 46.7% colorectal cancer samples compared with NCTs. The overexpression of miR-202-3p inhibited colorectal cancer cell growth in vitro and repressed tumorigenesis in nude mice. Then, miR-202-3p downregulated ADP-ribosylation factor-like 5A (ARL5A) protein level by binding to its 3' untranslated region, and knockdown of ARL5A phenocopied the proliferation inhibition effect of miR-202-3p. Furthermore, both of ARL5A mRNA and protein levels were upregulated in colorectal cancer samples compared with NCTs and high ARL5A protein levels predicted a poor prognosis. miR-202-3p might function as a tumor suppressor in colorectal cancer, and ARL5A, the functional target of miR-202-3p in colorectal cancer, is a potential prognostic factor for colorectal cancer.

Roles of MiR-101 and its Target Gene Cox-2 in Early Diagnosis of Cervical Cancer in Uygur Women

Early diagnosis is important for cervical cancer treatment. This study aimed to characteriz the microRNA profile and target gene protein levels of cervical cancers in Uygur women for application in early diagnosis. The profiles of miRNA in cervical cancer and chronic cervicitis were analyzed with miRNAmicroarray V4.0. The expression of miR-101 was detected by real-time PCR and locked nucleotide acid in situ hybridization (LNA-ISH). Cox-2 protein levels were assessed by immunohistochemistry. The microarray identified a set of 12 miRNAs significantly decreased in cervical cancer in comparison to the control group. Quantitative RT-PCR analysis showed miR-101 to be significantly downregulated in cancer tissues (p<0.05) while LNA-ISH showed miR-101 positive rates of 80% (20/25) and 8% (5/25) (p<0.05) in the control and cervical cancer groups. Cox-2 positive rates of cervical cancer and control groups were 84% (21/25) and 8% (2/25) (p<0.05). Use of down-regulation of miR-101 and up-regulation of Cox-2 as markers may play a role in early diagnosis of cervical cancer in Uygur women.

The microRNA-218 and ROBO-1 signaling axis correlates with the lymphatic metastasis of pancreatic cancer

Pancreatic cancer is known for its poor prognosis and early lymphatic metastasis is a notable characteristic. microRNAs (miRNAs) have been shown to be involved in the initiation and progression of pancreatic cancer. We, therefore, established a screening strategy to find miRNAs related to the lymphatic metastasis of pancreatic cancer and explored the target genes of miRNAs. miRNA array profiles were analyzed in tissue samples [pancreatic ductal adenocarcinoma (PDAC) and matched adjacent benign tissues (MAT)] and cell lines (BxPC-3-LN and BxPC-3). Combined analysis of profiling data from tissue samples and cell lines was used to identify miRNAs related to the lymphatic metastasis of pancreatic cancer. The expression levels of miRNAs were confirmed by real‑time reverse transcription PCR (RT-PCR) in tissue samples and cell lines. The correlation between miRNAs and clinicopathological characteristics was investigated. The expression features of miRNAs in pancreatic cancer, precursor lesions and metastatic lymph nodes were characterized by in situ hybridization (ISH). Predicted target genes of miRNAs were validated by RT-PCR and the protein levels of target genes were revealed by western blotting. Seventy and 63 miRNAs were differentially expressed in pancreatic cancer and BxPC-3-LN, compared to MAT and BxPC-3, respectively. Combined microarray analysis found 4 co-differentially expressed miRNAs (miRNA-663, miRNA-145, miRNA-218 and let-7) related to the lymphatic metastasis of pancreatic cancer. miRNA-218 was significantly downregulated in BxPC-3-LN (fold-change>10) and the expression levels of miRNA-218 were confirmed by RT-PCR. The group with lymph node metastasis and the elder group (age>64) showed lower expression of miRNA-218 (P=0.003 and 0.002), compared to patients without lymph nodes metastasis and patients in the younger group (age≤64), respectively. The expression of miRNA‑218 showed a decreasing trend from normal acinar/ductal epithelium, intraductal papillary mucinous neoplasm (IPMN), pancreatic cancer to metastatic lymph nodes by ISH. Among 8 predicted target genes of miRNA-218, rodent bone (ROBO-1) was confirmed to be upregulated in both mRNA and protein levels in pancreatic cancer. In conclusion, we established a screening strategy based on microarray results and found miRNA-218 to be a notable gene related to lymphatic metastasis of pancreatic cancer. Downregulation of miRNA-218 and upregulation of ROBO-1 were first demonstrated in pancreatic cancer. The miRNA-218 and ROBO-1 signaling axis may contribute to the lymphatic metastasis of pancreatic cancer.

Identification of microRNA activity by Targets' Reverse EXpression

Motivation: Non-coding microRNAs (miRNAs) act as regulators of global protein output. While their major effect is on protein levels of target genes, it has been proven that they also specifically impact on the messenger RNA level of targets. Prominent interest in miRNAs strongly motivates the need for increasing the options available to detect their cellular activity.Results: We used the effect of miRNAs over their targets for the detection of miRNA activity using mRNAs expression profiles. Here we describe the method, called T-REX (from Targets' Reverse EXpression), compare it to other similar applications, show its effectiveness and apply it to build activity maps. We used six different target predictions from each of four algorithms: TargetScan, PicTar, DIANA-microT and DIANA Union. First, we proved the sensitivity and specificity of our technique in miRNA over-expression and knock-out animal models. Then, we used whole transcriptome data from acute myeloid leukemia to show that we could identify critical miRNAs in a real life, complex, clinically relevant dataset. Finally, we studied 66 different cellular conditions to confirm and extend the current knowledge on the role of miRNAs in cellular physiology and in cancer.Availability: Software is available at http://aqua.unife.it and is free for all users with no login requirement.Contact: s.volinia@unife.itSupplementary information: Supplementary data are available at Bioinformatics online.

Stochastic model of transcription factor-regulated gene expression

We consider a stochastic model of transcription factor (TF)-regulated gene expression. The model describes two genes, gene A and gene B, which synthesize the TFs and the target gene proteins, respectively. We show through analytic calculations that the TF fluctuations have a significant effect on the distribution of the target gene protein levels when the mean TF level falls in the highest sensitive region of the dose–response curve. We further study the effect of reducing the copy number of gene A from two to one. The enhanced TF fluctuations yield results different from those in the deterministic case. The probability that the target gene protein level exceeds a threshold value is calculated with the knowledge of the probability density functions associated with the TF and target gene protein levels. Numerical simulation results for a more detailed stochastic model are shown to be in agreement with those obtained through analytic calculations. The relevance of these results in the context of the genetic disorder haploinsufficiency is pointed out. Some experimental observations on the haploinsufficiency of the tumour suppressor gene, Nkx 3.1, are explained with the help of the stochastic model of TF-regulated gene expression.

MDM2: A Novel Mineralocorticoid-Responsive Gene Involved in Aldosterone-Induced Human Vascular Structural Remodeling

Aldosterone has been demonstrated to play an important role in the pathogenesis of various cardiovascular diseases. Vascular structural remodeling, including vascular smooth muscle cell (VSMC) proliferation, has been also reported in small resistance arteries of patients with primary aldosteronism. Therefore, in this study, we examined whether genes involved in the regulation of the cell cycle were induced by aldosterone alone in cultured human VSMCs and in human small resistance arteries. Results of these studies eventually demonstrated that MDM2, one of the genes involved in anti-apoptosis and cell growth, was markedly increased in mineralocorticoid receptor (MR)-positive VSMCs by aldosterone in all microarray, reverse transcriptase-polymerase chain reaction, immunoblotting, and immunofluorescence analyses. In addition, an analysis using small interfering RNA demonstrated that this gene product was involved in cell proliferation of VSMCs induced by aldosterone. Eplerenone, a specific MR antagonist, inhibited this gene induction by aldosterone in VSMCs. MDM2 protein was also more abundant in VSMCs of small resistance arteries in patients with primary aldosteronism compared with a control population. MDM2 is therefore considered one of the mineralocorticoid-responsive genes that regulates cell proliferation of VSMCs induced by MR-mediated aldosterone stimulation, possibly playing an important role in aldosterone-induced vascular structural remodeling.

Expression of neurogranin and neuromodulin is affected in the striatum of vitamin A-deprived rats

Our previous data showed that vitamin A deficiency (VAD) induces, in whole brain, a reduced amount of mRNA for brain retinoic acid (RA) and triiodothyronine (T3) nuclear receptors (i.e., RAR, RXR, and TR, respectively), which is accompanied by reduced amounts of mRNA and protein of neurogranin (RC3, a neuronal protein involved in synaptic plasticity) as well as selective behavioral impairment. Given the important role of retinoids for optimal brain functioning, the effects of vitamin A depletion and subsequent administration of RA or T3 on the mRNA levels of RA and T3 nuclear receptors and on two target genes' ( RC3 and neuromodulin or GAP43) mRNA and protein levels were examined in the hippocampus, striatum, and cerebral cortex. A quantitative real-time polymerase chain reaction (PCR), in situ hybridization, and Western blot analysis demonstrated that the striatal region is the brain site where both RA and T3 signaling pathways are most affected by VAD. Indeed, rats fed a vitamin A-free diet for 10 weeks exhibited decreased expression of RAR, RXR, TR, RC3, and GAP43 in the striatum. The administration of T3 to these vitamin A-deprived rats reversed the reduction in mRNA levels of RA and T3 nuclear receptors and in mRNA and protein levels of target genes in this region. These data suggest that modifications that appear preferentially in the striatum, a region highly sensitive to vitamin A bioavailability, may contribute to neurobiological alterations and the spatial learning impairment that occurs in vitamin A-deprived animals.

Regulation of GAP-43 expression by chronic desipramine treatment in rat cultured hippocampal cells

Background The importance of molecular and cellular changes in hippocampus in major depression and in the mechanism of action of antidepressants has become increasingly clear. Identification of novel targets for antidepressants in hippocampus is important to understanding their therapeutic effects. Methods We used cDNA microarray to measure the expression patterns of multiple genes in primary cultured rat hippocampal cells. In situ hybridization and Northern and immunoblotting analysis were used to determine brain regional distribution and mRNA and protein levels of target genes. Results After comparing hybridized signals between control and desipramine treated groups, we found that chronic treatment with desipramine increased the expression of six genes and decreased the expression of two genes. One of the upregulated genes is growth associated protein GAP-43. In situ hybridization revealed that desipramine increased GAP-43 gene expression in dentate gyrus but not other brain regions. Northern and immunoblotting analysis revealed that desipramine increased GAP-43 mRNA and protein levels. GAP-43 expression is also increased by another antidepressant, tranylcypromine, but not by lithium or haloperidol. Conclusions Because GAP-43 regulates growth of axons and modulates the formation of new connections, our findings suggest that desipramine may have an effect on neuronal plasticity in the central nervous system.