1,725,425 research outputs found
Additional file 1 of miR-3189-targeted GLUT3 repression by HDAC2 knockdown inhibits glioblastoma tumorigenesis through regulating glucose metabolism and proliferation
No full textAdditional file 1: Supplementary Fig. 1. A Expression of Class 1 HDAC Genes and Proteins in GBM cells. GBM cells were extracted in lysis buffer, and cell lysates were analyzed using western blot analysis with class I HDAC antibodies (HDAC1, 2, 3 and 8). B Quantification of protein level of class I HDAC expression. Densitometric quantification of protein signals was quantified by ImageJ (Java 1.8.0_112, NIH, Bethesda, MD, USA), and the level of protein expression was normalized to β-actin. Data represent the means ± SD from three independent experiments. C Lentiviral infection of HDAC2 shRNA in GBM cells. A172 and U87MG cells were incubated for 6 days post-infection prior to measurement of cell viability by using MTT assay. All data are expressed as the mean ± SD for triplicates. D Class I HDAC siRNA was transfected into GBM cells. Cell lysates were analyzed by western blot using the indicated antibodies. E Class I HDAC siRNA was transfected into GBM and normal brain cells. SVGp12 and GBM cells were incubated for 48 h. Cell viability was measured via MTT assay. F qRT-PCR analysis and Immunoblot analysis of HDAC2 and GLUT3 expression in control GBM cells and HDAC2KD GBM cells upon doxycycline treatment. G IF in DOX-inducible shcontrol GBM cells and DOX-inducible shHDAC2 GBM cells upon doxycycline treatment (2.5 μg/ml) (DAPI: blue and FITC-HDAC2: green). Scale bar: 100 μm. H Luciferase reporter activities of DOX-inducible control and DOX-inducible shHDAC2 GBM cells (Upper: U87MG and Bottom: A172) with doxycycline. Cells were transiently transfected with reporter pGL3-Luc or pGL3-Puma-Luc plasmids. Dual luciferase activity was measured (420 nm) in cell lysates. Reporter activities were normalized relative to Renilla luciferase activities. I Cell viability of DOX-inducible shcontrol and DOX-inducible shHDAC2 GBM cells (Left: U87MG and Right: A172). GBM cells upon doxycycline treatment were measured in the presence or absence of Romidepsin by using WST-8 assay. All data are expressed as the mean ± SD from three independent experiments, each performed in triplicate. ***p < 0.001. Supplementary Fig. 2. A IF analysis of GLUT3 expression in DOX-induced shHDAC2 GBM cells and control GBM cells (DAPI: nuclei, Texas Red: GLUT3). Scale bar: 100 μm. B Kaplan-meier analysis of the Freije, Vital and Gravendeel dataset for SLC2A3 (GLUT3) expression. (P = 0.05). mRNA expression of GLUT3 and miR-3189 in control and HDAC2 siRNA-transfected GBM cells. C mRNA expression of GLUT3 and miR-3189 in control and HDAC2 siRNA-transfected GBM cells. D HDAC2 expression in Orthotropic Brain Tumor Mouse Models. Body weight of orthotropic xenograft mouse brain tumor models (DOX-inducible shControl U87MG and DOX-inducible shHDAC2 U87MG) with or without doxycycline treatment. E IHC staining of apoptotic cell death markers (Apaf-1 and Bax) in normal tissues and GBM tissues of mouse brain. F HDAC2 expression was measured in DOX-inducible shControl U87MG cells and DOX-inducible shHDAC2 U87MG cells orthotopically injected into mouse brains. Mice were given doxycycline (2 μg/ml) in drinking water. Supplementary Fig. 3. Quantitative PCR Analysis of mRNA Expression of GLUT3 and Cell Death Markers in Both GLUT3 siRNA- and miR-3189-transfected GBM Cells. A mRNA expression of GLUT3 by qRT-PCR in GLUT3 siRNA and miR-3189 mimic-transfected GBM cells. B mRNA expression of apoptotic cell death markers by qRT-PCR in control and GLUT3 siRNA and miR-3189 mimic-transfected GBM cells. All Data are expressed as the mean ± SD for triplicates. *p < 0.05, **p < 0.01, ***p < 0.001. Supplementary Fig. 4. Knockdown HDAC2 and GLUT3 Inhibits Glucose Metabolism and Cell Proliferation in GBM Cells. A Glucose uptake assay in GBM cells transfected with control siRNA or HDAC2 siRNA. B Metabolite measurement of DOX-inducible shHDAC2 GBM cells with or without doxycycline treatment. Left: Glucose uptake assay, Right: Lactate production assay. C Cell proliferation in HDAC2 siRNA-, GLUT3 siRNA-, or miR-3189-transfected GBM cells. Left: A172 cells, Right: U87MG cells. D Glucose uptake assay of GBM cells that were transfected with either GLUT1 siRNA or GLUT2 siRNA. Left: U87MG cells, Right: A172 cells. E Lactate production assay of GBM cells that were transfected with either GLUT1 siRNA or GLUT2 siRNA. Left: U87MG cells, Right: A172 cells. All data are expressed as the mean ± SD for triplicates. **p < 0.01, ***p < 0.001. Supplementary Fig. 5. Quantitative PCR Analysis of Selected miRNA in miR-3189-transfected GSCs. A HDAC2 mRNA expression in miR-3189-transfected GSCs (GSC20, GSC23, GSC28 and GSC267) after incubation for 48 h. Expression of HDAC2 mRNA was analyzed using qRT-PCR. B Expression of selected miRNAs by microarray analysis in miR-3189-transfected GSCs after incubation for 48 h. Expression of miRNAs was analyzed using qRT-PCR. All data are expressed as the mean ± SD for triplicates
Anti-tumoral effects of miR-3189-3p in glioblastoma
Get PDFGlioblastoma is one of the most aggressive brain tumors. We have previously found up-regulation of growth differentiation factor 15 (GDF15) in glioblastoma cells treated with the anticancer agent fenofibrate. Sequence analysis of GDF15 revealed the presence of a microRNA, miR-3189, in the single intron. We then asked whether miR-3189 was expressed in clinical samples and whether it was functional in glioblastoma cells. We found that expression of miR-3189-3p was down-regulated in astrocytoma and glioblastoma clinical samples compared with control brain tissue. In vitro, the functionality of miR-3189-3p was tested by RNA-binding protein immunoprecipitation, and miR-3189-3p coimmunoprecipitated with Argonaute 2 together with two of its major predicted gene targets, the SF3B2 splicing factor and the guanine nucleotide exchange factor p63RhoGEF. Overexpression of miR-3189-3p resulted in a significant inhibition of cell proliferation and migration through direct targeting of SF3B2 and p63RhoGEF, respectively. Interestingly, miR-3189-3p levels were increased by treatment of glioblastoma cells with fenofibrate, a lipid-lowering drug with multiple anticancer activities. The attenuated expression of miR-3189-3p in clinical samples paralleled the elevated expression of SF3B2, which could contribute to the activation of SF3B2 growth-promoting pathways in these tumors. Finally, miR-3189-3p-mediated inhibition of tumor growth in vivo further supported the function of this microRNA as a tumor suppressor
Influence of Carbon in Iron on Characteristics of Surface Modification by EDM in Liquid Nitrogen
Get PDFMany surface modification technologies have been proposed and carried out practically by CVD, PVD et.al. Carbonized layer has been made using EDM method. In this paper, to make the nitride layer by EDM some new trials were carried out using a titanium electrode in liquid nitrogen. Experiments were carried out on carbon steel (S45C), pure iron and cast iron. TiN can be obtained on EDMed surface. Moreover, TiCN can be found on cast iron and steel (S45C) by XRD investigation. To confirm the fabrication mechanisms of modified layer on the steel, the following experimental factors were investigated by EDS
Quisenberry, Frank E., 1892-1985 (SC 3189)
Get PDFFinding aid only for Manuscripts Small Collection 3189. Bound record book of Frank E. Quisenberry, Bowling Green, Kentucky, containing genealogical data on members of the Quisenberry and related families. Loose items included
Mechanisms of miR-3189-3p-mediated inhibition of c-MYC translation in triple negative breast cancer
Get PDFBACKGROUND: Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer characterized by the lack of estrogen receptor, progesterone receptor, and HER2. Our lab previously characterized miR-3189-3p as a microRNA with potent anti-cancer activity against glioblastoma. Here, we hypothesized a similar activity in TNBC cells. As miR-3189-3p is predicted to target a variety of RNA binding proteins, we further hypothesized an inhibitory effect of this miRNA on protein synthesis. METHODS: MDA-MB-231 and MDA-MB-468 cells were used to investigate the effect of miR-3189-3p on cell proliferation, migration, and invasion. TGCA database was used to analyze the expression of miR-3189-3p, c-MYC, 4EPB1, and eIF4E in breast cancer. Western blotting and RT-qPCR assays were used to assess the expression of selected proteins and RNAs after transfections. RESULTS: Although c-MYC is not a predicted gene target for miR-3189-3p, we discovered that c-MYC protein is downregulated in miRNA-treated TNBC cells. We found that the downregulation of c-MYC by miR-3189-3p occurs in both normal growth conditions and in the absence of serum. The mechanism involved the direct inhibition of eIF4EBP1 by miR-3189-3p. Additionally, we found that miR-3189-3p could negatively affect cap-independent translation mediated by internal ribosome entry sites (IRES) or by m6A. Finally, miR-3189-3p sensitized TNBC cells to doxorubicin. CONCLUSION: Overall, results indicated that miR-3189-3p exerts its anti-tumor activity through targeting translational regulatory proteins leading to an impairment in c-MYC translation, and possibly other oncogenic factors, suggesting that miR-3189-3p, alone or in combination, could be a valuable therapeutic approach against a malignancy with few treatment options
ANTI-TUMORAL EFFECTS OF MIR-3189-3P IN GLIOBLASTOMA
Get PDFIntroduzione: Il glioblastoma e’ un tumore maligno del cervello, della categoria dei gliomi, caratterizzato da rapida crescita cellulare, elevata invasivita’, aggressivita’ e resistenza alla radio e chemioterapia. I soggetti colpiti da questa forma tumorale, che costituisce circa il 50% delle neoplasie maligne cerebrali negli adulti, hanno in media un’aspettativa di vita di quindici mesi. Il trattamento di base prevede l’asportazione della massa tumorale in combinazione alla radioterapia, eventualmente seguite da chemioterapia con temozolomide. Data l’estrema necessita’ di sviluppare nuove strategie terapeutiche per questo tumore fino ad ora praticamente incurabile, l’individuazione dei meccanismi molecolari che sono alla base della proliferazione e della sopravvivenza del glioblastoma e’ di fondamentale importanza.
Growth Differentiation Factor 15 (GDF15), conosciuto anche con il nome di Nonsteroidal Anti-Inflammatory drug-activated Gene-1 (NAG-1) o Macrophage Inhibitory Cytokine-1 (MIC-1) e’ una proteina della famiglia del transforming growth factor-β (TGF-β). Il gene che codifica per GDF15 e’ costituito da due esoni che generano un precursore composto da un pro-peptide di 167 amminoacidi e da un dominio corrispondente al peptide maturo di 112 amminoacidi. In seguito alla dimerizzazione, avviene il clivaggio del precursore con il successivo rilascio del peptide maturo di 112 amminoacidi nella matrice extracellulare, dove agisce come dimero biologicamente attivo. L’espressione di GDF15 nei tessuti e’ notevolmente indotta in risposta a stimolazione con differenti farmaci anti-infiammatori, agenti citotossici, agonisti dei recettori della famiglia dei peroxisome proliferator-activated receptors (PPAR) e farmaci antitumorali.
Il ruolo di GDF15 nella tumorigenesi non e’ stato ancora del tutto chiarito. Evidenze sperimentali dimostrano che elevati livelli di trascritto sono presenti in pazienti durante la progressione dello stadio tumorale da astrocitoma a glioblastoma, ma altri studi hanno dimostrato come l’espressione di GDF15 sia indotta in risposta a trattamenti chemioterapici.
Metodi: L’espressione di GDF15 mediante saggi di Real-time PCR, Western Blot ed Elisa e del miR-3189-3p mediante Real-time PCR e’ stata valutata nella linea cellulare di glioblastoma LN-229 in seguito a stimolazione con Fenofibrato. Saggi di transfezione in vitro sono stati effettuati allo scopo di validare il targeting del miR-3189-3p sulla regione 3’UTR dei due geni SF3B2 e p63RhoGEF. Analisi di Real-time PCR, Western Blot, di proliferazione e migrazione cellulare in vitro e l’iniezione subcutanea ed intracranica in topi nudi di cellule di glioblastoma precedentemente transfettate con il miR-3189-3p, sono state eseguite al fine di studiare il ruolo funzionale del miR-3189-3p.
Risultati: Risultati precedentemente ottenuti nel nostro laboratorio hanno dimostrato che la stimolazione della linea cellulare LN-229 con il fenofibrato, un agonista di PPARα, determina un aumento di espressione di GDF15.
Nel presente studio, dall’analisi della sequenza genica di GDF15 e’ risultata di particolare interesse la presenza di un microRNA, miR-3189, all’interno del suo unico introne, in posizione prossimale all’esone 1. Il pre-mir-3189 contiene due sequenze di microRNA mature all’interno dell sua struttura a forcina: miR-3189-3p e miR-3189-5p, rispettivamente di 21 e 25 nucleotidi. Ad oggi non ci sono studi che abbiano riportato la funzione biologica di tale microRNA, pertanto dato il ruolo che ricoprono i microRNA nella regolazione genica, scopo principale di questo lavoro e’ stato innanzitutto quello di definire gli effetti dei due microRNA, miR-3189-3p e -5p, nella funzione biologica di GDF15 nei glioblastomi.
Abbiamo scoperto che l’induzione del trascritto e della proteina in seguito a stimolazione con fenofibrato e’ accompagnata da una elevata espressione del miR-3189-3p e precede gli eventi di apoptosi innescati dal fenofibrato. Nelle medesime condizioni sperimentali non e’ stata osservata invece induzione del miR-3189-5p. Inoltre, mediante saggi di transfezione, abbiamo dimostrato che l’espressione ectopica del miR-3189-3p determina una inibizione della proliferazione e della migrazione cellulare mediante il silenziamento di due dei suoi mRNA bersaglio predetti, rispettivamente il fattore di splicing SF3B2 e il fattore di scambio del nucleotide guanina p63RhoGEF.
Dall’analisi di espressione genica su campioni di glioblastoma e di tessuti normali abbiamo trovato che ad un aumento dei livelli di espressione di GDF15 nei glioblastomi corrisponde un decremento dei livelli di miR-3189-3p e, in aggiunta, queste differenze di espressione correlano con un incremento nei livelli di SF3B2 e una tendenza all’aumento in p63RhoGEF.
Infine, esperimenti di iniezione subcutanea e intracranica di cellule di glioblastoma precedentemente transfettate con il miR-3189-3p, hanno mostrato una inibizione della crescita tumorale rispetto a cellule di controllo.
Conclusioni: Tutte queste evidenze sperimentali supportano e validano il ruolo di miR-3189-3p come oncosoppressore nei glioblastomi mediante il controllo della crescita e della migrazione cellulare attraverso il silenziamento rispettivamente di SF3B2 e p63RhoGEF.SUMMARY
Background: Glioblastoma is a deadly cancer characterized by rapid cell proliferation, high invasiveness, and resistance to radio- and chemotherapy. Patients with this aggressive tumor, which accounts for nearly 50% of all adult brain tumors, have a median survival of approximately 15 months. The standard treatment for glioblastoma involves invasive surgery and radiotherapy, which is often followed by chemotherapy with temolozomide. As the development of novel therapeutic treatments for glioblastoma are desperately needed, it is essential to understand the molecular mechanisms supporting growth and survival of this highly malignant and practically incurable brain tumor.
Growth Differentiation Factor 15, GDF15, also known as Nonsteroidal Anti-inflammatory drug-activated Gene -1 (NAG-1) or Macrophage Inhibitory Cytokine-1 (MIC-1), is a member of the transforming growth factor-β (TGF-β) superfamily. The GDF15 gene is encoded by two exons to generate a precursor protein containing a 167 amino acid pro-peptide sequence and a 112 amino acid mature domain. Upon dimerization, this precursor protein is cleaved resulting in the release of the 112 amino acid mature GDF15 peptide, which is secreted into the extracellular matrix as a biologically active dimer. GDF15 can be induced by anti-inflammatory drugs, cytotoxic agents, peroxisome proliferator-activated receptor (PPAR) agonist, and anticancer drugs. Increased GDF15 mRNA expression has been reported in patients during malignant progression to glioblastoma, and others have reported that expression levels of GDF15 are upregulated in glioblastoma cells in response to cytotoxic stimuli during chemotherapy treatment. We have previously reported increased expression of GDF15 in the LN-229 glioblastoma cell line following the treatment with fenofibrate, an agonist of PPARα. In this study, we have analyzed the genomic sequence of GDF15 and found it contains a microRNA, miR-3189, encoded within its single intron at a position proximal to exon 1. The precursor sequence encoded by miR-3189 contains two mature microRNA sequences within the stem-loop: miR-3189-3p and miR-3189-5p, of 21 and 25 nucleotides in length, respectively. The biological function of this microRNA has never been described before. Because of the role of microRNAs in gene regulation, we wanted to define the effects of these co-expressed microRNAs, miR-3189-3p and miR-3189-5p, in the biological function of GDF15.
Methods: The expression of GDF15 by Real-time PCR, Western Blots and Elisa assays and miR-3189-3p by Real-time PCR was evaluated in LN-229 cells stimulated with Fenofibrate. Transfections were performed in order to validate the targeting of miR-3189-3p on the 3’UTR of two of its major predicted targets SF3B2 and p63RhoGEF. The functional role of miR-3189-3p was assessed through Real-time PCR, Western Blot, cell-growth and migration assays, and through both the subcutaneous and the intracranial injection in nude mice.
Results: We found that treatment of glioblastoma cells with fenofibrate resulted in a striking increase in GDF15 mRNA and protein levels, which was accompanied by high expression of miR-3189-3p, and preceded fenofibrate-induced apoptosis. In this experimental condition, miR-3189-5p was not detected. Ectopic expression of miR-3189-3p inhibited LN-229 cell growth and migration through downregulation of the splicing factor SF3B2 and the guanine nucleotide exchange factor p63RhoGEF, respectively. In comparison to the normal brain tissue, we also found that glioblastoma clinical samples have increased levels of GDF15 and decreased levels of miR-3189-3p, and that these changes correlated with increased expression of SF3B2 and a trend of increased levels for p63RhoGEF. Finally, both the subcutaneous and the intracranial growth of glioblastoma cells expressing miR-3189-3p were significantly reduced when compared to control cells, thus further validating the role of this microRNA as a tumor suppressor.
Conclusions: Our studies have demonstrated that miR-3189-3p has a tumor-suppressive role by controlling the growth and the migration of glioblastoma cells by targeting the SF3B2 and p63RhoGEF mRNAs
MECHANISMS OF MIR-3189-3P-MEDIATED ANTITUMORAL ACTIVITY IN BREAST CANCER
Get PDFBackground: Brest cancer is one of the most diffused tumors worldwide. In US, breast cancer represents 30% of all newly diagnosed cancers in female and remain the second leading cause of death, with new 281,550 diagnosed cases in women. Invasive breast cancer can be divided into four subtypes, among which HR+ is the most common and the less aggressive. On the contrary, TNBC is the most aggressive subtype and the one with the worst prognosis. TNBC is characterized by the lack of estrogen receptor, progesterone receptor, and HER2. Consequently, TNBC cannot be treated with the available hormone therapies and receptor targeted treatments. Currently, even if some TNBCs do benefit from immunotherapy and PARP inhibitors, surgery and chemotherapy appear to remain the first-line treatments.
The regulatory oncoprotein c-MYC regulate several pathways and is generally overexpressed in many tumors including TNBCs, making it a promising therapeutic target. Nevertheless, c-MYC is currently considered a non-drugable target.
MiRNAs are small endogenous ncRNAs, 18-25 bp long, that are able to regulate gene expression and our lab previously characterized miR-3189-3p as a miRNA with a strong anticancer activity against different tumor types, including glioblastoma and melanoma. Because of the role demonstrated in these tumors, we wanted to investigate the effect of miR-3189-3p in breast cancer.
Methods: in this work, we used MDA-MB-231 cell line transiently transfected with miR-3189-3p or scramble control. The functional role of the miRNA was assessed by cell proliferation, cell migration and cell invasion assays. The miR-3189-3p gene target 4E-BP1 3’UTR was validated by luciferase assays. MiRNA-mediated c-MYC downregulation was assessed by Real time PCR and Western Blots. The role of 4E-BP1 in the miRNA-mediated effects was assessed through its overexpression or downregulation. The role of miR-3189-3p on the metabolism was assessed using the Seahorse XF96 Flux Analyzer.
Results: We demonstrated that overexpression of miR-3189-3p reduces cell proliferation, migration and invasion. Additionally, although MYC 3’UTR is not a predicted target for miR-3189-3p, we discovered that our miRNA is able to downregulate c-MYC protein levels by targeting 4E-BP1 in normal growth conditions and in stress conditions. Indeed, miR-3189-3p has a negative effect on cap-dependent and cap-independent translation. Finally, we found that miR-transfected cells show a different metabolism at their basal state, increasing both oxygen metabolism and glycolysis.
Conclusion: Overall, results indicated that miR-3189-3p targets translational regulatory proteins that negatively affect expression of c-MYC, suggesting that miR-3189-3p can be a valuable therapeutic approach against a malignancy with few treatment options
El proceso de impugnación de acuerdos en la casación n° 3189 – 2012. Lima Norte
No full textEl objetivo de esta investigación fue: Analizar la vía procesal para impugnar los acuerdos celebrados en una asociación en el marco de la Casación N° 3189 – 2012. Lima Norte.
La investigación fue de tipo descriptivo. El diseño usual de la Indagación fue el no experimental y el diseño específico fue el descriptivo transversal por que se trata de estudiar
La población estuvo delimitada por la Casación N° 3189-2012 Lima Norte. El escenario cultural se desenvuelve dentro del ámbito del Derecho Civil, de acuerdo a la jurisprudencia vinculante Corte Suprema de Justicia de la República. Quinto Pleno Casatorio Civil, siendo la muestra de estudio la Casación N° 3189-2012 Lima Norte.
La Técnica a utilizar será el análisis documental. El instrumento que se empleó en la recolección de la información es la Casación N° 3189-2012 Lima Norte.Trabajo de suficiencia profesiona
- …
