51 research outputs found
Expression of urinary miRNAs targeting NLRs inflammasomes in bladder cancer
No full textAim of the study: Inflammasome, a large complex of NOD-like receptors (NLRs), drives tumor growth and progression. The present study aimed at exploring the alteration in expression of urinary inflammasome-related microRNAs (miRNAs) in bladder cancer (BC). Our previous report demonstrated the up-regulation of NLRs genes (NLRP3, NLRP4, NLRP9 and NAIP) in urine sediments of patients harboring BC. The expression levels of miRNAs targeting these NLRs (miR-146a-5p, miR-106a-5p, miR-17-5p, miR-223-3p, miR-141-3p, miR-19a-3p, miR-145-5p, miR-185-5p) were assayed in the same patient cohort. Materials and methods: Forty-six subjects affected by BC, 28 healthy controls (CTR0) and 31 subjects with histologically confirmed bladder inflammation (CTR1) were recruited. Total RNA was extracted from urine sediment and resulting cDNA was used for amplification by real-time polymerase chain reaction. MiRNA expression levels were evaluated and compared among selected groups. Patients were further stratified according to tumor stage, grade and risk of recurrence and progression. Moreover, non-muscle invasive low-grade and high-grade (HG) BC patients were compared. Results: MiR 141-3p and miR-19a-3p expression decreased in CTR1 with respect to both BC and CTR0. In contrast, miR-146a-5p was up-regulated in BC compared with CTR0. MiR106a-5p, miR17-5p and miR19a-5p were significantly up-regulated in HG, high-risk (HR) and non-muscle invasive HG BC patients, while miR-185-5p was significantly higher in muscle invasive tumors, according to T stage stratification. Conclusion: The increased expression of miRNAs targeting NLRs in HG and HR BC patients is in accordance with the decrease in NLR mRNAs observed in our previous report. These data corroborate the direct role of NLR genes and respective regulatory miRNAs in BC making these inflammasome-related molecules a reliable non-invasive tool for BC diagnosis
Stability assessment of candidate reference genes in urine sediment of prostate cancer patients for miRNA applications
No full textWe aimed at assessing the stability of candidate reference genes in urine sediments of men subjected to digital rectal examination for suspected prostate cancer (PCa). Two microRNAs (miR-191 and miR-25) and 1 small nucleolar RNA (SNORD48) were assayed in 35 post-DRE urine sediments of men with PCa and in 26 subjects with histologically confirmed benign prostatic hyperplasia (BPH). The stability of candidate reference genes was assessed through BestKeeper algorithm and equivalence test. miR-200b and miR-452 were used to test for the effect of normalization on target genes. Our results proved miR-191 to be the most stable gene, showing the lowest degree of variation and the highest stability value. miR-25 and SNORD48 values fell beyond the cutoff of acceptability. In conclusion, we recommend the use of miR-191 for normalization purposes in post-DRE urine sediments
Antisense to Epstein Barr virus-encoded LMP1 does not affect the transcription of viral and cellular proliferation-related genes, but induces phenotypic effects on EBV-transformed B lymphocytes
No full textIt is generally accepted that Epstein-Barr virus (EBV) latent genes EBNA-2, EBNA-3A, -3C, EBNA-LP and LMP1 are essential for growth transformation and immortalization of B lymphocytes. Among these genes, LMP1 plays a key role in the survival and dissemination of the infected B cells by inducing anti-apoptotic genes and surface expression of several activation antigens and adhesion molecules. We have previously shown that antisense oligodeoxynucleotides directed to LMP1 mRNA, effectively suppress LMP1 gene expression and substantially reduce B95.8 cell proliferation. In this study, we have used antisense LMP1 oligomers to investigate whether LMP1 suppression might influence the expression of latent EBV genes with oncogenic potential, anti-apoptotic genes, or affect the phenotype of EBV-infected B95.8 cells. Our data show that LMP1 suppression does not affect the transcription of EBNA-2, EBNA-3A, -3B and -3C genes, or that of bcl-2 and mcl-1 anti-apoptotic genes. In contrast, consistent modifications in the expression of CD39, CD54, CD23, CD11 and CD10 molecules were observed in B95.8 cells after treatment with antisense LMP1. Our findings support the possibility for using LMP1 antisense oligomers as therapeutics in EBV-associated tumors
Characterization of Kallireins and microRNAs in Urine Sediment for the Discrimination of Prostate Cancer from Benign Prostatic Hyperplasia.
No full textProstate Cancer (PCa) and Benign Prostatic Hyperplasia (BPH) are frequently coexisting in elderly men. The measurement of serum PSA together with Digital Rectal Examination (DRE) represents the primary diagnostic tool to suspect PCa, whereas definitive diagnosis is achieved by prostate biopsy. The low specificity of PSA and the modest detection rate of biopsy convict the patient to a quite often unnecessary and uncomfortable clinical itinerary. There is a urgent need for new and more accurate methodologies to diagnosize PCa. In the present study, the expression of 4 mRNAs and 2 miRNAs was evaluated in post DRE urine cell pellets from patients suffering PCa and age-matched subjects affected by BPH with elevated PSA levels. We also evaluated the diagnostic accuracy of markers in predicting PCa
Interaction of EBV latent origin of replication with the nuclear matrix:identificatio of S/MAR sequences and protein components
No full textAbstractDuring latency, Epstein Barr virus (EBV) genome, as an episome, is attached to the nuclear matrix (NM) via the latent origin of replication ori P. Within this element, we have found that a region, 580 bp long, encompassing the replicator DS element, shows the strongest affinity for the NM. In addition, by cross-linking with cis-diamminedichloroplatinum, we have identified two NM proteins with an apparent molecular weight of 85 and 60 kDa that, with high affinity and specificity, bind ori P. These proteins are not induced by EBV infection, but their interaction with ori P is lost upon induction of EBV lytic cycle. These data strongly suggest that the binding of ori P to specific components of the NM is required for EBV latent replication
Sequence specific visualization of DNA in live mammalian cells
No full textIn the first part of this work, the fate of plasmid DNA labeled with PNA was
investigated in living cells. It was shown that foreign DNA rapidly undergoes
interaction with intranuclear structural sites that strongly reduce its mobility
and restrict the DNA to regions excluding nucleoli and nuclear bodies as PML
bodies. The labeled plasmids partially colocalize with SAF-A, a well characterized
marker of the nuclear matrix, and are resistant towards extraction by detergent
and, in part, elevated salt concentration. Importantly the localization and low
mobility are independent of plasmid sequence and do not require the presence
of either a S/MAR DNA element or a functional promoter.
In the second part of this work, the attention was focused on chromatin
mobility in living mammalian cells by using the lac operator/repressor system.
The experiments show that the labeled chromatin loci are highly dynamic,
moving around by random walk, but are locally constrained in a defined nuclear
volume. Interestingly, the mobility of the analyzed loci depends on their location
and partially on the transcriptional status of the cell
Inhibition of Poly(ADP-ribose)polymerase impairs Epstein Barr Virus lytic cycle progression
No full textAbstract Background Poly(ADP-ribosylation) is a post-translational modification of nuclear proteins involved in several cellular events as well as in processes that characterize the infective cycle of some viruses. In the present study, we investigated the role of poly(ADP-ribosylation) on Epstein-Barr Virus (EBV) lytic cycle activation. Results Inhibition of PARP-1 by 3-aminobenzamide (3-ABA) during EBV induction, diminished cell damage and apoptosis in the non-productive Raji cell line while markedly reducing the release of viral particles in the productive Jijoye cells. Furthermore, incubation with 3-ABA up-regulated the levels of LMP1 and EBNA2 latent viral proteins. At the same time, it slightly affected the expression of the immediate early BZLF1 gene, but largely down-regulated the levels of the early BFRF1 protein. The modulation of the expression of both latent and lytic EBV genes appeared to be post-transcriptionally regulated. Conclusion Taken together the data indicate that PARP-1 plays a role in the progression of EBV lytic cycle and therefore, PARP inhibitors might represent suitable pharmacological adjuncts to control viral spread in EBV productive infection.</p
BSE-associated prion-amyloid cardiomyopathy in primates
No full textPrion amyloidosis occurred in the heart of 1 of 3 macaques intraperitoneally inoculated with bovine spongiform encephalopathy prions. This macaque had a remarkably long duration of disease and signs of cardiac distress. Variant Creutzfeldt-Jakob disease, caused by transmission of bovine spongiform encephalopathy to humans, may manifest with cardiac symptoms from prion-amyloid cardiomyopathy
Localization and dynamics of small circular DNA in live mammalian nuclei
No full textWhile genomic DNA, packaged into chromatin, is known to be locally constrained but highly dynamic in the nuclei of living cells, little is known about the localization and dynamics of small circular DNA molecules that invade cells by virus infection, appli-cation of gene therapy vectors or experimental transfection. To address this point, we have created traceable model substrates by direct labeling of plasmid DNA with ¯uorescent peptide nucleic acids, and have investigated their fate after microinjection into living cells. Here, we report that foreign DNA rapidly undergoes interactions with intranuclear structural sites that strongly reduce its mobility and restrict the DNA to regions excluding nucleoli and nuclear bodies such as PML bodies. The labeled plasmids partially co-localize with SAF-A, a well characterized marker protein for the nuclear `scaf-fold ' or `matrix', and are resistant towards extraction by detergent and, in part, elevated salt concentra-tions. We show that the localization and the low mobility of plasmids is independent of the plasmid sequence, and does not require the presence of either a scaffold attachment region (SAR) DNA element or a functional promoter
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