Análisis de la expresión del GEN IGF-I identificado como oncogén en células de glioma de ratón parentales y transfectadas usando estrategia de terapia génica por triple hélice y anti sentido.

Abstract

Cancer is one of the diseases that has generated the highest mortality rate in recent times, being glioblastoma the most common brain cancer, between 2 to 19 cases / 100,000 inhabitants per year have been found in the last decade in Colombia (WHO) [1], this type of cancer corresponds to a primary tumor of the central nervous system (CNS) and occurs in the glial cells located on the sides of the neurons, various investigations indicate that it originates from an alteration in the hormonal signaling routes that regulate cell proliferation and survival causing uncontrolled division that originates the formation of tumors and inhibits programmed cell death, the alterations can be caused by acquired or inherited genetic mutations. Different studies of this type of cancer have determined that an increase in the level of expression of the gene that encodes the protein called Insulin-Like Growth Factor type I (IGF-I) is related to the appearance of glioblastoma, being considered as an oncogene. The IGF-I protein is similar to insulin in function and structure, and is a member of the family of proteins that are involved in mediating cell growth and development. Several strategies have been used to treat this disease, but no significant impact has been found on the treatment of this type of cancer or on the life expectancy of patients. Therefore, the aim of this study was to use antisense and triple helix gene therapy to regulate the expression levels of the IGF-I gene in glioblastoma-type cancer at the Genetics and Chronicles Laboratory of the National Institute of Health and the Biochemistry and Molecular Biology Research Group of the Francisco José de Caldas District University. For this, somatic cells of the mouse glioma cell line C6 were used, which were cultured and transfected by lipofection method with the expression vectors pAnti – IGF-I and pMT-EP that had inserted respectively a 540 bp sequence (antisense) and a 23 bp sequence (triple helix). To evaluate the expression levels of the IGF-I gene of the cells, mRNA extraction, quantification and analysis by RT-PCR were performed before and after being subjected to treatment. Also, a fragment of the IGF-I gene from the samples of the mouse glioma cell line C6, frontal cancer and malignant brain tumor (glioma) were sequenced by capillary electrophoresis to determine if there were differences between their sequences with respect to a normal control (white blood cell DNA). The optimal medium for the growth of glioblastoma-type cells was found to be DMEM - Glutamax, for transfection the optimal amount of vector - lipofectamine was found to be 1.5 uL (1: 1 ratio), it was also observed that the time of complex formation between lipofectamine and expression vector was 20 minutes and the optimal exposure time of the cells for the complexes to enter the cytosol was 6 hours. The quantitative real-time PCR study using taqman probes specific for IGF-I cDNA demonstrated a decrease in gene expression of 80% when triple helix therapy was used, compared to the antisense strategy where a 60% inhibition in gene expression was observed. When comparing the sequence of the IGF-I gene fragment from samples from the C6 mouse glioma cell line, frontal cancer, and malignant brain tumor (glioma), no mutations were found, indicating that this type of cell only presents alterations in the IGF-I gene in its degree of expression and not in its sequence. These findings are promising and allow us to propose new research in human cells in vitro and in vivo so that in the future they can be used in humans through the use of cell therapy with the purpose of obtaining vaccines to reduce mortality from this type of cancer that currently constitutes a public health problem worldwide.