Evaluation of in vitro functional role of microRNAs in Cervical Cancer progression

Abstract

Cervical Cancer is the most important HPV-induced disease, as it is a serious public health problem worldwide. Morbidity and mortality from Cervical Cancer have tended to decline over the past 30 years in many countries because of the effectiveness of widespread implementation of prevention programs. However, the prognosis and 5-years survival rate of patients with advanced Cervical Cancer remains poor. To date, there is no specific molecular marker capable of predicting the progression of Cervical Cancer, especially biomarkers involving samples from minimally invasive collection, such as Liquid-Based Cytology (LBC). Therefore, the identification of new prognostic markers is as important as characterizing the underlying molecular mechanisms at the onset and development of Cervical Cancer. In this mode, microRNAs (miRNAs) are promising molecules, as recent studies have shown that besides regulating the expression of key genes for the development and progression of Cancer they are specific tissue. with this, the aim of this study is to investigate the in vitro role of microRNAs (hsa-miR-205-5p, hsa-miR-130a-3p, hsa-miR-4531 and hsa-miR-381-3p) identified in an earlier study of the group and already reported in the literature with an important role in carcinogenesis, through functional assays in commercial cell lines and primary Cervical Cancer. Initially the expression of miRNAs will be evaluated by real-time PCR (qPCR) in the panel of commercial and primary strains, in order to select the cell lines for the in vitro assays. Later, ectopic expression of the miRNAs of interest (hsa-miR-4531 and hsa-miR-381-3p) will be performed by overexpression and / or silencing in selected cell lines. In order to evaluate the impact of in vitro interventions, we will evaluate the proliferation, migration, cell invasion and apoptosis capacity in the selected strains. Finally, we will evaluate the gene expression profile of the strains (before and after ectopic expression) using the NanoString technology using PanCancer Pathways panel in order to establish a miRNA-mRNA interaction network and to identify the main targets of these miRNAs differentially expressed in cell lines . Validation of target genes for these miRNAs will be done by the in vitro luciferase assay. We believe that the evaluation of the role of these in vitro miRNAs identified as potential biomarkers of Cervical Cancer precursor lesions may better elucidate the molecular mechanisms of Cervical Cancer initiation and progression. (AU)