Identification and characterization of microRNAs involved in inhibition of the NF-kB signaling pathway by the action of glycyrrhizinate dipotassium in glioblastoma multiforme cell lines

Abstract

Glioblastoma multiforme (GBM) accounts for 15-20% of neurological tumors and being considered the most aggressive. Biological heterogeneity and resistance to chemotherapy are the major obstacles facing the development of effective treatment for GBM. Several genes and signaling pathways have been identified to be responsible for the aggressive phenotype of this tumor. The NF-ºB pathway is continuously active in GBM tumor cells by regulating elevated expression of anti-apoptotic and cell survival genes, adhesion and invasion factors. Glycyrrhizic acid (GA) has been shown to reduce the inflammatory process by suppressing the expression of the nuclear protein NF-ºB p65 and to increase expression of the p53 tumor suppressor, caspase-9, and cleaved caspase-9 pro-apoptotic proteins. Glycyrrhizinate dipotassium (DPG) is a byproduct of GA and also has anti-inflammatory properties, but without the side effects observed with oral administration of GA. Previous assays in our lab, with GBM cell lines, U87MG and T98G, demonstrated that DPG have cytotoxic and antiproliferative effect. DPG treatment was also found to induce apoptosis in cell lines using DNA fragmentation assay and cleaved caspase-3 pro-apoptotic protein expression. In addition, DPG inhibited the NF-ºB pathway by modulating the microRNAs (miRs) mir-16 and miR-146a, which inhibited the expression of their target genes IRAK2 and TRAF6, respectively. The primary endpoint of this study is to evaluate the inhibitory effect of DPG on the NF-ºB pathway by the modulation of miRs. For an identification of miRs, an overall analysis of 96 miRs, previously known as regulators of genes involved with NF-ºB pathway, will be performed using U87MG cell line treated with DPG. Next, we will select a group of miRs differentially expressed in DPG-treated U87MG cell line to be validated by qPCR in other GBM cell lines, U87MG, T98G, U138MG and U251 exposed to DPG. In addition, the effect that DPG exerts on the cell cycle and cell death by flow cytometry, TUNEL assay and western blotting for caspase-9 and PARP will be evaluated, to evaluate DPG as a therapeutic target for GBM. (AU)