The MicroRNA Family Gets Wider: The IsomiRs Classification and Role

MicroRNAs (miRNAs or miRs) are the most characterized class of non-coding RNAs and are engaged in many cellular processes, including cell differentiation, development, and homeostasis. MicroRNA dysregulation was observed in several diseases, cancer included. Epitranscriptomics is a branch of epigenomics that embraces all RNA modifications occurring after DNA transcription and RNA synthesis and involving coding and non-coding RNAs. The development of new high-throughput technologies, especially deep RNA sequencing, has facilitated the discovery of miRNA isoforms (named isomiRs) resulting from RNA modifications mediated by enzymes, such as deaminases and exonucleases, and differing from the canonical ones in length, sequence, or both. In this review, we summarize the distinct classes of isomiRs, their regulation and biogenesis, and the active role of these newly discovered molecules in cancer and other diseases

A new monoclonal antibody detects downregulation of protein tyrosine phosphatase receptor type γ in chronic myeloid leukemia patients

Background: Protein tyrosine phosphatase receptor gamma (PTPRG) is a ubiquitously expressed member of the protein tyrosine phosphatase family known to act as a tumor suppressor gene in many different neoplasms with mechanisms of inactivation including mutations and methylation of CpG islands in the promoter region. Although a critical role in human hematopoiesis and an oncosuppressor role in chronic myeloid leukemia (CML) have been reported, only one polyclonal antibody (named chPTPRG) has been described as capable of recognizing the native antigen of this phosphatase by flow cytometry. Protein biomarkers of CML have not yet found applications in the clinic, and in this study, we have analyzed a group of newly diagnosed CML patients before and after treatment. The aim of this work was to characterize and exploit a newly developed murine monoclonal antibody specific for the PTPRG extracellular domain (named TPγ B9-2) to better define PTPRG protein downregulation in CML patients. Methods: TPγ B9-2 specifically recognizes PTPRG (both human and murine) by flow cytometry, western blotting, immunoprecipitation, and immunohistochemistry. Results: Co-localization experiments performed with both anti-PTPRG antibodies identified the presence of isoforms and confirmed protein downregulation at diagnosis in the Philadelphia-positive myeloid lineage (including CD34+/CD38bright/dim cells). After effective tyrosine kinase inhibitor (TKI) treatment, its expression recovered in tandem with the return of Philadelphia-negative hematopoiesis. Of note, PTPRG mRNA levels remain unchanged in tyrosine kinase inhibitors (TKI) non-responder patients, confirming that downregulation selectively occurs in primary CML cells. Conclusions: The availability of this unique antibody permits its evaluation for clinical application including the support for diagnosis and follow-up of these disorders. Evaluation of PTPRG as a potential therapeutic target is also facilitated by the availability of a specific reagent capable to specifically detect its target in various experimental conditions

Regulative Loop between \u3b2-catenin and Protein Tyrosine Receptor Type \u3b3 in Chronic Myeloid Leukemia

Protein tyrosine phosphatase receptor type \u3b3 (PTPRG) is a tumor suppressor gene, down-regulated in Chronic Myeloid Leukemia (CML) cells by the hypermethylation of its promoter region. \u3b2-catenin (CTNNB1) is a critical regulator of Leukemic Stem Cells (LSC) maintenance and CML proliferation. This study aims to demonstrate the antagonistic regulation between \u3b2-catenin and PTPRG in CML cells. The specific inhibition of PTPRG increases the activation state of BCR-ABL1 and modulates the expression of the BCR-ABL1- downstream gene \u3b2-Catenin. PTPRG was found to be capable of dephosphorylating \u3b2-catenin, eventually causing its cytosolic destabilization and degradation in cells expressing PTPRG. Furthermore, we demonstrated that the increased expression of \u3b2-catenin in PTPRG-negative CML cell lines correlates with DNA (cytosine-5)-methyl transferase 1 (DNMT1) over-expression, which is responsible for PTPRG promoter hypermethylation, while its inhibition or down-regulation correlates with PTPRG re-expression. We finally confirmed the role of PTPRG in regulating BCR-ABL1 and \u3b2-catenin phosphorylation in primary human CML samples. We describe here, for the first time, the existence of a regulative loop occurring between PTPRG and \u3b2-catenin, whose reciprocal imbalance affects the proliferation kinetics of CML cells

Gene expression landscape of Chronic Myeloid Leukemia K562 cells overexpressing the tumor suppressor gene PTPRG

This study concerns the analysis of the modulation of Chronic Myeloid Leukemia (CML) cell model K562 transcriptome following transfection with the tumor suppressor gene encoding for Protein Tyrosine Phosphatase Receptor Type G (PTPRG) and treatment with the tyrosine kinase inhibitor (TKI) Imatinib. Specifically, we aimed at identifying genes whose level of expression is altered by PTPRG modulation and Imatinib concentration. Statistical tests as differential expression analysis (DEA) supported by gene set enrichment analysis (GSEA) and modern methods of ontological term analysis are presented along with some results of current interest for forthcoming experimental research in the field of the transcriptomic landscape of CML. In particular, we present two methods that differ in the order of the analysis steps. After a gene selection based on fold-change value thresholding, we applied statistical tests to select differentially expressed genes. Therefore, we applied two different methods on the set of differentially expressed genes. With the first method (Method 1), we implemented GSEA, followed by the identification of transcription factors. With the second method (Method 2), we first selected the transcription factors from the set of differentially expressed genes and implemented GSEA on this set. Method 1 is a standard method commonly used in this type of analysis, while Method 2 is unconventional and is motivated by the intention to identify transcription factors more specifically involved in biological processes relevant to the CML condition. Both methods have been equipped in ontological knowledge mining and word cloud analysis, as elements of novelty in our analytical procedure. Data analysis identified RARG and CD36 as a potential PTPRG up-regulated genes, suggesting a possible induction of cell differentiation toward an erithromyeloid phenotype. The prediction was confirmed at the mRNA and protein level, further validating the approach and identifying a new molecular mechanism of tumor suppression governed by PTPRG in a CML context

Regulative loop between \u3b2-Catenin and Protein Tyrosine Phosphatase Receptor Type \u3b3 (PTPRG) in Chronic Myeloid Leukemia

La Leucemia Mieloide Cronica (CML) \ue8 una malattia mieloproliferativa caratterizzata dalla presenza di BCR-ABL1, una tirosin chinasi coinvolta nell\u2019attivazione aberrante di vie molecolari responsabili dei processi di carcinogenesi legati a questa malattia. PTPRG (tirosin fosfatasi recettoriale gamma) \ue8 un gene onco soppressore la cui espressione nella Leucemia Mieloide Cronica \ue8 negativamente regolata dall\u2019ipermetilazione del suo promotore. Studi precedenti hanno dimostrato che l\u2019espressione indotta della proteina PTPRG \ue8 correlata ad una ridotta formazione di colonie ed ad una diminuita capacit\ue0 clonogenica delle cellule leucemiche. Inoltre, un ripristino della sua espressione \ue8 stato osservato nei pazienti dopo la terapia con inibitori specifici contro le tirosin chinasi. Al fine di comprendere la regolazione reciproca tra questa fosfatasi e BCR-ABL1, abbiamo cercato possibili interattori molecolari per PTPRG tra le proteine appartenenti a vie molecolari attivate da BCR-ABL1 e ci siamo concentrati sullo studio della proteina \u3b2-Catenina (CTNNB1), che rappresenta allo stesso tempo un target di PTPRG ed un suo regolatore trascrizionale. Abbiamo osservato che una modulazione di espressione e funzione di PTPRG in linee cellulari di CML porta a cambiamenti nella fosforilazione e nell'attivazione di BCR-ABL1 e della \u3b2-Catenina: PTPRG \ue8 in grado di legare e defosforilare l'onco-proteina \u3b2-Catenina provocando la sua destabilizzazione nell\u2019ambiente citosolico con conseguente proteolisi in cellule con una espressione esogena o endogena di PTPRG (linee cellulari K562 e LAMA-84). Inoltre, abbiamo dimostrato che l\u2019attivit\ue0 trascrizionale della \u3b2-Catenina in linee cellulari che non esprimono PTPRG provoca un\u2019aumentata espressione del gene DNA (citosina-5) \u2013metiltransferasi 1 (DNMT1), responsabile dell\u2019ipermetilazione del promotore di PTPRG, e che il blocco della sua attivit\ue0 dopo il trattamento con un inibitore delle metiltransferasi (Azacitidina) ed il silenziamento di questo gene con uno specifico siRNA sono correlati ad una ripristinata espressione di PTPRG, sia a livello trascrizionale che proteico. In questo progetto dimostriamo per la prima volta un meccanismo che coinvolge la degradazione della \u3b2-Catenina, mediata dalla defosforilazione ad opera della fosfatasi PTPRG, e la conseguente inibizione trascrizionale dei geni regolati dal complesso di trascrizione TCF4 / \u3b2-Catenina. Da parte sua, anche la \u3b2-Catenina regola PTPRG dal momento che \ue8 in grado di attivare la trascrizione di DNMT1, che contribuisce alla metilazione del promotore di PTPRG, impedendone la corretta trascrizione. Abbiamo ipotizzato l\u2019esistenza di un loop regolativo tra PTPRG e la \u3b2-Catenina dimostrando che uno squilibrio del sistema a favore di una o l'altra potrebbe determinare un diverso destino proliferativo per le cellule di Leucemia Mieloide Cronica e per la loro aggressivit\ue0 clinica.Chronic Myeloid Leukemia (CML) is a myeloproliferative disease characterized by the presence of BCR-ABL1 tyrosine kinase. PTPRG (Protein Tyrosine Phoshatase Receptor type \u3b3) is a tumor suppressor gene down-regulated by hypermethylation of its promoter region in CML. Previous studies demonstrated that a re-expression of PTPRG protein is correlated with a decreased colony formation and clonogenic capability of CML cells. In addition, its restored expression was observed in patients after TKI therapy. In order to understand the mutual regulation between this phosphatase and BCR-ABL1, we searched for PTPRG putative interactors among proteins that are downstream BCR-ABL1 driven pathways and we focused on \u3b2-Catenin (CTNNB1), that resulted at the same time a PTPRG substrate and its transcriptional regulator. We observed that a modulation of expression and function of PTPRG in CML cell lines leads to changes in phosphorylation and activation of BCR-ABL1 and \u3b2-Catenin: PTPRG is able to bind and dephosphorylate the onco-protein \u3b2-Catenin causing its cytosolic destabilization and consequent degradation in cells with an exogenous or endogenous expression of PTPRG (K562 and LAMA-84 cell lines). Furthermore, we demonstrated that an increased expression of \u3b2-Catenin in PTPRG negative CML cell lines is correlated with an over-expression of the DNA (cytosine-5)-methyltransferase 1 (DNMT1) that is responsible of PTPRG promoter hypermethylation and that inhibition after treatment with 5-Azacydine and down-regulation of this gene are correlated with PTPRG re-expression both at mRNA and protein levels. Here we show for the first time a mechanism that involves \u3b2-Catenin degradation and the consequent down-regulation of genes regulated by the TCF4/\u3b2-Catenin transcription complex. In return, \u3b2-Catenin up-regulation is correlated with an over-expression of DNMT1 that contributes to hypermethylation of PTPRG promoter region. We hypothesized that there is a regulative loop between PTPRG and \u3b2-Catenin and that an imbalance of the system in favor of one or the other could determine a different proliferation fate of CML cells and their clinical aggressiveness

Identification of protein tyrosine phosphatase receptor gamma extracellular domain (sPTPRG) as a natural soluble protein in plasma.

PTPRG is a widely expressed protein tyrosine phosphatase present in various isoforms. Peptides from its extracellular domain have been detected in plasma by proteomic techniques. We aim at characterizing the plasmatic PTPRG (sPTPRG) form and to identify its source.The expression of sPTPRG was evaluated in human plasma and murine plasma and tissues by immunoprecipitation and Western blotting. The polypeptides identified have an apparent Mr of about 120 kDa (major band) and 90 kDa (minor band) respectively. Full length PTPRG was identified in the 100.000×g pelleted plasma fraction, suggesting that it was present associated to cell-derived vesicles (exosomes). The release of sPTPRG by HepG2 human hepatocellular carcinoma cell line was induced by ethanol and sensitive to metalloproteinase and not to Furin inhibitors. Finally, increased levels of the plasmatic ∼120 kDa isoform were associated with the occurrence of liver damage.These results demonstrate that sPTPRG represent a novel candidate protein biomarker in plasma whose increased expression is associated to hepatocyte damage. This observation could open a new avenue of investigation in this challenging field

Detecting and Characterizing A-To-I microRNA Editing in Cancer

Adenosine to inosine (A-to-I) editing consists of an RNA modification where single adenosines along the RNA sequence are converted into inosines. Such a biochemical transformation is catalyzed by enzymes belonging to the family of adenosine deaminases acting on RNA (ADARs) and occurs either co- or post-transcriptionally. The employment of powerful, high-throughput detection methods has recently revealed that A-to-I editing widely occurs in non-coding RNAs, including microRNAs (miRNAs). MiRNAs are a class of small regulatory non-coding RNAs (ncRNAs) acting as translation inhibitors, known to exert relevant roles in controlling cell cycle, proliferation, and cancer development. Indeed, a growing number of recent researches have evidenced the importance of miRNA editing in cancer biology by exploiting various detection and validation methods. Herein, we briefly overview early and currently available A-to-I miRNA editing detection and validation methods and discuss the significance of A-to-I miRNA editing in human cancer

Expression and release of PTPRG by HepG2 cells.

<p><b>Panel A</b>: WB with Rb anti-P4 of total lysate and supernatant of HepG2 cell line. Lane 1: 10 μg total cell lysate of HepG2 cell line. Lane 2: serum-free conditioned medium of HepG2 after 100000×g ultra-centrifugation and TCA precipitation. Black arrow: full-length protein, dashed arrow: ∼120 kDa isoform, gray arrow: ∼90 kDa isoform. <b>Panel B</b>: WB with Rb anti-P4 of serum-free conditioned medium of HepG2 cells showing down regulation of sPTPRG by siRNA (siRNA PTPRG) in comparison with a scrambled sequence (SCR). Coomassie blue staining of serum-free conditioned samples demonstrating the loading on comparable amounts of material in both lanes. <b>Panel C</b>: serum-free conditioned medium (SFCM) of HepG2 cells. Left: Molecular weight marker. NT: untreated cells, DMSO: cells treated with vehicle (DMSO), or overnight with 12 μM metalloproteinase inhibitor GM6001 (Ilomastat). Densitometric analysis of sPTPRG 120 kDa isoform related to a common aspecific band approximately at 70 kDa present in all samples (<b>below</b>). <b>Panel D</b>: cells treated overnight with 50 μM furin inhibitor. Dashed arrow: ∼120 kDa isoform. Below is the densitometric analysis expressed as fold increase versus control of the ∼120 kDa band normalized against the total protein load. WB was performed with a chicken anti-PTPRG antibody[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119110#pone.0119110.ref027" target="_blank">27</a>]. <b>Panel E</b>: WB with streptavidin-HRP of cell-surface biotinylated HepG2 cells untreated (NT) or treated (EtOH) for 16 hours with 50 mM ethanol, lysed and immunoprecipitated with anti-P4 antibody (IP/WB, bands boxed). Lower levels of full length PTPRG is present in EtOH treated cells. The same antibody was used in WB analysis on the corresponding serum-free conditioned media precipitated with TCA/acetone (right panel, boxed). The ∼120 kDa PTPRG isoform was detectable in SFCM at higher levels in comparison with untreated cells. The first two lanes represent total cell lysate before IP and demonstrate equal amount of protein and comparable surface biotin labeling of the two samples.</p

Localization of PTPRG-derived peptides.

<p>Schematic representation of peptides identified by large-scale proteome analysis and reported in the literature[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119110#pone.0119110.ref020" target="_blank">20</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119110#pone.0119110.ref022" target="_blank">22</a>]. In grey are the sequences found in all the studies. ECD: extracellular domain, ICD: intracellular domain.</p