Functional validation of microRNA target genes in hepatocellular carcinoma.

Author(s)

Carolina Fazio Campos

• Document type: Master's Dissertation
• Press: Botucatu. 2020-09-28.
• Institution: Universidade Estadual Paulista (Unesp). Faculdade de Medicina. Botucatu
• Defense date: 2020-08-26
• Advisor: Patricia Pintor dos Reis

Abstract

Introduction: MicroRNAS (miRNAs) are small non-coding RNAs that and regulate the expression of genes associated with several biological mechanisms. miRNAs have been indicated as potential biomarkers for the diagnosis, prognosis and treatment of several types of cancer, including hepatocellular carcinoma (HCC). HCC is the fourth most common cause of cancer death and is often associated with poor prognosis, aggression, drug resistance and advanced diagnosis. Aim: To identify and experimentally validate the interaction between miRNA/target genes related to the HCC tumorigenesis process and contribute to the identification of molecular pathways relevant to tumor development. In addition, to verify the effect of changing the expression of the chosen miRNA in the cell proliferation, migration and invasion of the liver cancer cell lines. Material and Methods: The selection of miRNA was performed based on the analysis of previous data of our group and bioinformatic analyses. The identification of the target genes was performed using computational prediction tools (mirDIP and miRTarBase). The HCC cell lines (Hep 3B and PCL/PRF/5) were transfected with the selected miRNA mimic or negative control, in order to evaluate the expression of target genes (RT-qPCR). After transfection, the potential of cell proliferation (MTS), migration (wound healing) and invasion (transwell assay) of the cell lines was evaluated. Luciferase assay was used to confirm miRNA binding to the target gene. Results: miR-451a was selected for functional validation by being consistently detected as underexpressed in HCC from three databases (primary patient tumors, meta-analysis and TCGA). In silico analysis revealed 12 putative candidate genes regulated by miR-451a, relevant in HCC tumorigenesis and not yet experimentally validated: FMNL3, BUB1B, SPC25, KIF2A, PLEKHG2, BAK1, HMGB2, MEF2D, CDKN2B, HELLS, RRAGD and CSE1L. In both cell lines, the increase in miR-451a expression by transfection of the mimic miRNA resulted in a significant decrease (p <0.05) of SPC25 gene expression. The luciferase assay demonstrated the binding of the microRNA to the SPC25 gene, confirming that the SPC25 gene is a direct target of miR-451a. Furthermore, the restoration of miR-451a expression led to decrease in cell proliferation, migration and invasion of the Hep 3B and PCL/PRF/5 cell lines. Conclusion: The SPC25 gene was identified as a new target of miR-451a. Moreover, our findings indicate that miR-451a is related to HCC tumorigenesis and progression, being suggested as a potential target for HCC therapy. (AU)

• FAPESP's process: 18/06354-2 - Functional validation of microRNA target genes in hepatocellular carcinoma
• Grantee: Carolina Fazio Campos
• Support Opportunities: Scholarships in Brazil - Master