Functional validation of microRNA target genes in hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC) is the second most common cause of cancer death and the sixth most common cancer, worldwide. MicroRNAs (miRNAs) are small non-protein-coding RNAs and potent regulators of gene expression involved in relevant biological mechanisms. These mechanisms are associated with diseases such as cancer. miRNAs have been indicated as robust biomarkers for the diagnosis, prognosis and potential molecules for cancer treatment. Therefore, the purpose of this study is the functional validation of a specific miRNA, miR-451a, and two of its target genes (SPC25 and BUB1B) involved in the Rho GTPase pathway. These genes were selected from experimental data generated by our group, combined with data from a meta-analysis of the literature and bioinformatic data analysis. We propose to use the following methodologies: (a) the luciferase assay to experimentally validate whether miR-451a directly regulates target genes SPC25 and BUB1B and (b) analysis of the migration potential and invasion of HCC cell lines, before and after transfection with miRNA mimics for mir-451a, since this miRNA is significantly decreased in all analyzed data sets and in cell lines. Expression of miR-451a and target genes SPC25 and BUB1B will be evaluated by Reverse Transcription followed by Quantitative Real-Time PCR. This study will contribute to the generation of functional validation data of a miRNA and specific target genes in molecular pathways involved in tumorigenesis but not yet elucidated in HCC. Migration and cell invasion assays will be important to verify the effect of miR-451a and SPC25 and BUB1B target genes associated with HCC oncogenesis. Data generated in this research may contribute to better characterization of biomarkers with potential clinical application for HCC patients. (AU)