Uso de oligonucleótidos antisentido contra los RNA mitocondriales no codificantes en células de mieloma de ratón: efecto sobre la viabilidad y proliferación celular
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2010
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es
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Universidad Andrés Bello
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En el laboratorio de Cáncer de la Fundación Ciencia para la Vida, se ha descrito una
familia de RNAs mitocondriales no codificantes (ncmtRNA) que se expresan de manera
diferencial dependiendo del tipo celular y, permiten diferenciar una célula normal de una
tumoral. Esta familia está compuesta por dos moléculas, el RNA mitocondrial nocodificante
sentido (ncmtRNA-S) que es expresado tanto en células normales
proliferantes como en células tumorales, pero nó en células en reposo, y dos RNAs
mitocondriales antisentido (ncmtRNAs-AS 1 y 2) que se encuentran sobre expresados
en células normales proliferantes, pero reprimidos en células tumorales, al igual que en
células en reposo. Si bien aún no se ha dilucidado la función que estos ncmtRNAs
cumplen, la evidencia experimental muestra que al interferir los ncmtRNAs-AS con
oligonucleótidos complementarios, se induce muerte de células tumorales humanas. En
consecuencia, se indujo muerte en células de mieloma de ratón (NS0/2) como modelo
pre-clínico utilizando ODN-FPS antisentido contra los ncmtRNAs-AS in vitro, y se
analizó la vía implicada en la muerte celular. Células mesenquimáticas de médula ósea
de ratón (MSC) se usaron como células normales en proliferación. Células NS0/2 y
MSC fueron transfectadas con ODN-FPS complementarios a los ncmtRNA durante 24h.
El porcentaje de muerte fue determinado por tinción con azul tripán, ensayo de
morfología nuclear (DAPI), TUNEL y anexina V. En células NS0/2 el mayor efecto se
obtuvo con un oligonucleótido complementario al ncmtRNA-AS, en concentraciones de
50-100 nM, obteniéndose porcentajes de muerte de alrededor del 50%. El tratamiento
indujo fragmentación de DNA, exposición de fosfatidilserina y una fuerte disminución en
la proliferación celular. Sin embargo, células MSC no se vieron afectadas por el
tratamiento. Estos resultados muestran que la "terapia" con oligonucleótido antisentido
contra los ncmtRNAs-AS permite inducir una muerte por apoptosis selectiva para
células tumorales, lo que permitirá establecer un protocolo aplicable a estudios
preclínicos de eliminación ex vivo de células tumorales de médula ósea para
autotransplante.
At the cancer laboratory, in Fundación Ciencia para la Vida, a novel family of non-coding mitochondrial RNAs (ncmtRNAs) has been described. These RNAs express differentially according to the cell type, and allow for a normal cell to be distinguished from a tumor cell. In human cell lines, this family is composed of two molecules, the sense non-coding mitochondrial RNA (S-ncmtRNA) which is expressed both in normal proliferating and tumor cells, but not in resting cells, and the antisense non-coding mitochondrial RNAs (AS1 and AS2-ncmtcRNAs) which are also over expressed in normal proliferating cells, but are repressed in tumor and resting cells. Even though the function of these RNAs has not been described yet, experimental evidence shows that when the AS-ncmtRNAs are interfered with complementary oligonucleotides, death of human tumour cells is achieved. In consequence, death of mouse myeloma cells (NS0/2) as a pre-clinical model was induced in vitre, using phosphorotioate ODNs (FPS-ODN) against the antisense RNAs, to analyze de type of death that was triggered by it. Mouse bone marrow Mesenquimal cells (MBMMC) where used as a control of normal cells in proliferation. Both type of cells where transfected with complementary FPS-ODNs against the ncmtRNAs for 24 hours. The percentage of death was determined by trypan blue staining, nuclear morphology assay (DAPI), TUNEL and anexin V. We observed that in NS0/2 cells, the best effect was obtained using an ODN against the antisense transcprits at a concentration ranging from 50 to 1 OOnM, which showed a death percentage of 50%. The treatment induced DNA fragmentation, phosphatidilserine translocation and a strong downregulation of the cell proliferation. MSC on the other hand, were not affected by the treatment. These results show that the therapy using FPS-ODNs against the AS-ncmtRNAs, induces selective death by apoptosis of tumor cells. These results will allow to establish a methology for the application of the therapy in pre clinic studies for the removal of tumor cells in an ex vivo manner for autologous bone marrow transplantation.
At the cancer laboratory, in Fundación Ciencia para la Vida, a novel family of non-coding mitochondrial RNAs (ncmtRNAs) has been described. These RNAs express differentially according to the cell type, and allow for a normal cell to be distinguished from a tumor cell. In human cell lines, this family is composed of two molecules, the sense non-coding mitochondrial RNA (S-ncmtRNA) which is expressed both in normal proliferating and tumor cells, but not in resting cells, and the antisense non-coding mitochondrial RNAs (AS1 and AS2-ncmtcRNAs) which are also over expressed in normal proliferating cells, but are repressed in tumor and resting cells. Even though the function of these RNAs has not been described yet, experimental evidence shows that when the AS-ncmtRNAs are interfered with complementary oligonucleotides, death of human tumour cells is achieved. In consequence, death of mouse myeloma cells (NS0/2) as a pre-clinical model was induced in vitre, using phosphorotioate ODNs (FPS-ODN) against the antisense RNAs, to analyze de type of death that was triggered by it. Mouse bone marrow Mesenquimal cells (MBMMC) where used as a control of normal cells in proliferation. Both type of cells where transfected with complementary FPS-ODNs against the ncmtRNAs for 24 hours. The percentage of death was determined by trypan blue staining, nuclear morphology assay (DAPI), TUNEL and anexin V. We observed that in NS0/2 cells, the best effect was obtained using an ODN against the antisense transcprits at a concentration ranging from 50 to 1 OOnM, which showed a death percentage of 50%. The treatment induced DNA fragmentation, phosphatidilserine translocation and a strong downregulation of the cell proliferation. MSC on the other hand, were not affected by the treatment. These results show that the therapy using FPS-ODNs against the AS-ncmtRNAs, induces selective death by apoptosis of tumor cells. These results will allow to establish a methology for the application of the therapy in pre clinic studies for the removal of tumor cells in an ex vivo manner for autologous bone marrow transplantation.
Notas
Tesis (Magister en Biotecnología)
Palabras clave
Cáncer de Hueso, Ratas - Experimentos, Células Cancerosas