Examinando por Autor "Burzio, Luis O."
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Ítem Antisense noncoding mitochondrial RNA-2 gives rise to miR-4485-3p by Dicer processing in vitro(BioMed Central Ltd, 2021-12) Farfán, Nicole; Sanhueza, Nicole; Briones, Macarena; Burzio, Luis O.; Burzio, Verónica A.Background: The antisense noncoding mitochondrial RNAs (ASncmtRNAs) derive from the mitochondrial 16S gene. Knockdown of these transcripts with chemically-modified antisense oligonucleotides induces proliferative arrest, apoptosis and invasiveness reduction in tumor but not normal cells. One of these transcripts, ASncmtRNA-2, contains the complete and identical sequence of hsa-miR-4485-3p and, upon knockdown of this transcript, there is a strong increase in levels of this miRNA, suggesting ASncmtRNA-2 as a source for miR-4485-3p, which is supported by several evidences from our group and others, in the ex vivo setting. Results: Here we show that incubation of in vitro-transcribed ASncmtRNA-2 with recombinant Dicer produces RNA fragments corresponding to hsa-miR-4485-3p, showing that Dicer binds to and processes ASncmtRNA-2, strongly supporting the hypothesis that ASncmtRNA-2 acts as a precursor for miR-4485-3p. Conclusion: The in vitro results presented here strengthen the hypothesis that miR-4485-3p is derived from ASncmtRNA-2 by Dicer processing. Since miR-4485-3p is classified as a tumor suppressor miRNA, this evidence strengthens the application of ASncmtRNA knockdown for cancer therapy.Ítem Down-regulation of the antisense mitochondrial non-coding RNAs (ncRNAs) is a unique vulnerability of cancer cells and a potential target for cancer therapy(2014) Vidaurre, Soledad; Fitzpatrick, Christopher; Burzio, Verónica A.; Briones, Macarena; Villota, Claudio; Villegas, Jaime; Echenique, Javiera; Oliveira-Cruz, Luciana; Araya, Mariela; Borgna, Vincenzo; Socías, Teresa; Lopez, Constanza; Avila, Rodolfo; Burzio, Luis O.Hallmarks of cancer are fundamental principles involved in cancer progression. We propose an additional generalized hallmark of malignant transformation corresponding to the differential expression of a family of mitochondrial ncRNAs (ncmtRNAs) that comprises sense and antisense members, all of which contain stem-loop structures. Normal proliferating cells express sense (SncmtRNA) and antisense (ASncmtRNA) transcripts. In contrast, the ASncmtRNAs are down-regulated in tumor cells regardless of tissue of origin. Here we show that knockdown of the low copy number of the ASncmtRNAs in several tumor cell lines induces cell death by apoptosis without affecting the viability of normal cells. In addition, knockdown of ASncmtRNAs potentiates apoptotic cell death by inhibiting survivin expression, a member of the inhibitor of apoptosis (IAP) family. Down-regulation of survivin is at the translational level and is probably mediated by microRNAs generated by dicing of the double-stranded stem of the ASncmtRNAs, as suggested by evidence presented here, in which the ASncmtRNAs are bound to Dicer and knockdown of the ASncmtRNAs reduces reporter luciferase activity in a vector carrying the 3′-UTR of survivin mRNA. Taken together, down-regulation of the ASncmtRNAs constitutes a vulnerability or Achilles' heel of cancer cells, suggesting that the ASncmtRNAs are promising targets for cancer therapy. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.Ítem Expression of a novel non-coding mitochondrial RNA in human proliferating cells(Oxford University Press, 2007-12-01) Villegas, Jaime; Burzio, Veronica; Villota, Claudio; Landerer, Eduardo; Martinez, Ronny; Santander, Marcela; Martinez, Rodrigo; Pinto, Rodrigo; Vera, Maria I.; Boccardo, Enrique; Villa, Luisa L.; Burzio, Luis O.Previously, we reported the presence in mouse cells of a mitochondrial RNA which contains an inverted repeat (IR) of 121 nucleotides (nt) covalently linked to the 5 end of the mitochondrial 16S RNA (16S mtrRNA). Here, we report the structure of an equivalent transcript of 2374 nt which is over-expressed in human proliferating cells but not in resting cells. The transcript contains a hairpin structure comprising an IR of 815 nt linked to the 5 end of the 16S mtrRNA and forming a long double-stranded structure or stem and a loop of 40 nt. The stem is resistant to RNase A and can be detected and isolated after digestion with the enzyme. This novel transcript is a non-coding RNA (ncRNA) and several evidences suggest that the transcript is synthesized in mitochondria. The expression of this transcript can be induced in resting lymphocytes stimulated with phytohaemagglutinin (PHA). Moreover, aphidicolin treatment of DU145 cells reversibly blocks proliferation and expression of the transcript. If the drug is removed, the cells re-assume proliferation and over-express the ncmtRNA. These results suggest that the expression of the ncmtRNA correlates with the replicative state of the cell and it may play a role in cell proliferation.Ítem HPV-18 E2 protein downregulates antisense noncoding mitochondrial RNA-2, delaying replicative senescence of human keratinocytes(Aging, 2019-01-01) Villota, Claudio; Varas-Godoy, Manuel; Jeldes, Emanuel; Campos, América; Villegas, Jaime; Borgna, Vincenzo; Burzio, Luis O.; Burzio, Verónica A.Human and mouse cells display a differential expression pattern of a family of mitochondrial noncoding RNAs (ncmtRNAs), according to proliferative status. Normal proliferating and cancer cells express a sense ncmtRNA (SncmtRNA), which seems to be required for cell proliferation, and two antisense transcripts referred to as ASncmtRNA-1 and -2. Remarkably however, the ASncmtRNAs are downregulated in human and mouse cancer cells, including HeLa and SiHa cells, transformed with HPV-18 and HPV-16, respectively. HPV E2 protein is considered a tumor suppressor in the context of high-risk HPV-induced transformation and therefore, to explore the mechanisms involved in the downregulation of ASncmtRNAs during tumorigenesis, we studied human foreskin keratinocytes (HFK) transduced with lentiviral-encoded HPV-18 E2. Transduced cells displayed a significantly extended replicative lifespan of up to 23 population doublings, compared to 8 in control cells, together with downregulation of the ASncmtRNAs. At 26 population doublings, cells transduced with E2 were arrested at G2/M, together with downregulation of E2 and SncmtRNA and upregulation of ASncmtRNA-2. Our results suggest a role for high-risk HPV E2 protein in cellular immortalization. Additionally, we propose a new cellular phenotype according to the expression of the SncmtRNA and the ASncmtRNAs.Ítem Interferencia del ARN mitocondrial no codificante por lentivirus psuedotipificados(Universidad Andrés Bello, 2012) Varas Godoy, Manuel; Burzio, Luis O.; Valenzuela, PabloBurzio y colaboradores han descrito una familia de ARNs mitocondriales no codificantes (ARNmtnc) denominados ARNmtnc Sentido (ARNmtnc-S) y ARNmtnc Antisentido (ARNmtnc-AS) que se encuentran diferencialmente expresados en células normales y tumorales. Células normales en proliferación expresan altos niveles tanto del ARNmtnc-S como del ARNmtnc-AS. En contraste, células que no están en división expresan bajos niveles de ambos transcritos. Interesantemente, células tumorales expresan el ARNmtnc-S y disminuyen la expresión del ARNmtnc-S. Estas características son observadas tanto en células en cultivo como en biopsias de tejidos. Previamente, Burzio y colaboradores demostraron que la transfección transiente con oligonucleótidos complementarios a estos ARNmtnc induce muerte en diferentes tipos de células cancerígenas in vitro, pero no en células normales, dando la posibilidad de usar la interferencia de estos ARNs mitocondriales como un potencial tratamiento contra el cáncer. El objetivo de este estudio fue determinar si la administración in vivo de lentivirus que codifican shRNA (del inglés short hairpain RNA) dirigidos contra los ARNmtnc podría inhibir el crecimiento tumoral y la metástasis en modelos animales de melanoma de ratón. Para esto se construyeron vectores lentivirales no-replicativos que codifican un shRNA contra los ARNmtnc (Lv-ARNmtnc) y luciferasa (Lv-Control) y su efecto fue evaluado in vitro por transducción de células de melanoma de ratón B16F10. Mediciones por PCR en tiempo real indican una disminución de aproximadamente 6 veces en la expresión del ARNmtnc-S y del ARNmtnc-AS en células transducidas con Lv-ARNmtnc en comparación a células transducidas con Lv-Control. Además, la transducción de Lv-ARNmtnc induce muerte celular apoptótica determinada por exposición en la superficie celular de fosfatidilserina y fragmentación del ADN. La capacidad de estos lentivirus para inhibir el crecimiento de tumores sólidos y metástasis de células de melanoma B16F10 fue evaluada in vivo. Ratones C57BLl6 a los cuales se les indujeron tumores subcutáneos con células B16F10 fueron tratados cada dos días con cinco inyecciones intratumorales de 4 x lo7 Lv-ARNmtnc o LV-Control en un volumen de 100 VI. Los ratones tratados con Lv-ARNmtnc mostraron una significativa disminución en el volumen tumoral comparado a ratones tratados con LV-Control observado hasta el día 20 post inoculación de las células. Para el ensayo de metástasis, ratones C57BLl6 fueron inyectados por vía intravenosa con células B16F10 y cuatro días después se les administraron cada tres días 4 inyecciones intravenosas de 5 x 10' Lv-ARNmtnc o Lv-Control en un volumen de 200 pl. Los ratones fueron sacrificados dos semanas después de la inyección de las células para el análisis de metástasis pulmonar. Los ratones tratados con Lv-ARNmtnc mostraron una significativa reducción en el número de focos metastásicos comparados a los ratones tratados con Lv-Control y también una reducción en el peso pulmonar. Tinción de secciones de tejido con hematoxilina-eosina indica que los pulmones de los ratones tratados con Lv-ARNmtnc presentan solo unos pocos nódulos. En contraste, los nódulos metastásicos ocupaban la mayoría del tejido pulmonar cuando los ratones fueron tratados con Lv-Control. Estos resultados sugieren que la administración intratumoral y sistémica de shRNA dirigidos a los ARNmtnc representan una promisoria estrategia hacia el desarrollo de una potencial terapia contra el cáncer.Ítem Inverse Modulation of Aurora Kinase A and Topoisomerase IIα in Normal and Tumor Breast Cells upon Knockdown of Mitochondrial ASncmtRNA(MDPI, 2023-10) Bendek, Maximiliano F.; Fitzpatrick, Christopher; Jeldes, Emanuel; Boland, Anne; Deleuze, Jean-François; Farfán, Nicole; Villegas, Jaime; Nardocci, Gino; Montecino, Martín; Burzio, Luis O.; Burzio, Verónica A.Breast cancer is currently the most diagnosed form of cancer and the leading cause of death by cancer among females worldwide. We described the family of long non-coding mitochondrial RNAs (ncmtRNAs), comprised of sense (SncmtRNA) and antisense (ASncmtRNA) members. Knockdown of ASncmtRNAs using antisense oligonucleotides (ASOs) induces proliferative arrest and apoptotic death of tumor cells, but not normal cells, from various tissue origins. In order to study the mechanisms underlying this selectivity, in this study we performed RNAseq in MDA-MB-231 breast cancer cells transfected with ASncmtRNA-specific ASO or control-ASO, or left untransfected. Bioinformatic analysis yielded several differentially expressed cell-cycle-related genes, from which we selected Aurora kinase A (AURKA) and topoisomerase IIα (TOP2A) for RT-qPCR and western blot validation in MDA-MB-231 and MCF7 breast cancer cells, as well as normal breast epithelial cells (HMEC). We observed no clear differences regarding mRNA levels but both proteins were downregulated in tumor cells and upregulated in normal cells. Since these proteins play a role in genomic integrity, this inverse effect of ASncmtRNA knockdown could account for tumor cell downfall whilst protecting normal cells, suggesting this approach could be used for genomic protection under cancer treatment regimens or other scenarios.Ítem Mitochondrial ASncmtRNA-1 and ASncmtRNA-2 as potent targets to inhibit tumor growth and metastasis in the RenCa murine renal adenocarcinoma model(Impact Journals LLC, 2017) Borgna, Vincenzo; Villegas, Jaime; Burzio, Verónica A.; Belmar, Sebastián; Araya, Mariela; Jeldes, Emanuel; Lobos-González, Lorena; Silva, Verónica; Villota, Claudio; Oliveira-Cruz, Luciana; Lopez, Constanza; Socias, Teresa; Castillo, Octavio; Burzio, Luis O.Knockdown of antisense noncoding mitochondrial RNAs (ASncmtRNAs) induces apoptosis in several human and mouse tumor cell lines, but not normal cells, suggesting this approach for a selective therapy against different types of cancer. Here we show that in vitro knockdown of murine ASncmtRNAs induces apoptotic death of mouse renal adenocarcinoma RenCa cells, but not normal murine kidney epithelial cells. In a syngeneic subcutaneous RenCa model, treatment delayed and even reversed tumor growth. Since the subcutaneous model does not reflect the natural microenviroment of renal cancer, we used an orthotopic model of RenCa cells inoculated under the renal capsule. These studies showed inhibition of tumor growth and metastasis. Direct metastasis assessment by tail vein injection of RenCa cells also showed a drastic reduction in lung metastatic nodules. In vivo treatment reduces survivin, N-cadherin and P-cadherin levels, providing a molecular basis for metastasis inhibition. In consequence, the treatment significantly enhanced mouse survival in these models. Our results suggest that the ASncmtRNAs could be potent and selective targets for therapy against human renal cell carcinoma. © Borgna et al.