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Screening miR-450a and miR-450b targets in ovarian carcinoma cell lines A2780 and SKOV-3 by PAR-CLIP

Grant number: 16/22307-9
Support Opportunities:Scholarships abroad - Research Internship - Doctorate
Effective date (Start): April 01, 2017
Effective date (End): November 30, 2017
Field of knowledge:Biological Sciences - Genetics - Human and Medical Genetics
Principal Investigator:Wilson Araújo da Silva Junior
Grantee:Bruna Rodrigues Muys
Supervisor: Markus Hafner
Host Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
Research place: National Institutes of Health, Bethesda (NIH), United States  
Associated to the scholarship:13/25326-6 - Functional study of microRNAs miR-450a and miR-450b-5p in tumorigenesis, BP.DR


Ovarian cancer is the most lethal gynecologic cancer, and most patients have a survive rate of fewer than five years after diagnosis, due to lack of symptoms in its early stages. Therefore, there is an urgent need to develop biomarkers to help diagnose ovarian cancer earlier. MicroRNAs (miRNAs) are small noncoding RNA molecules that act regulating critical biological processes such as development, cell proliferation, differentiation, metabolism, and apoptosis. Many miRNAs are associated with tumorigenesis, and some of them have been found to operate as oncogenes or tumor suppressor genes. miRNAs play a major role in post-transcriptional gene regulation and suppress gene expression by guiding the so-called RNA-induced silencing complex to partially sequence complementary sites found mainly in the untranslated regions of target mRNAs. Data from studies conducted as part of my Ph.D. project revealed that the miRNAs miR-450a and miR-450b-5p were underexplored and predominately underexpressed in ovary tumor cell lines compared to normal correspondent tissues. The study also showed that both miRNAs impaired migration and invasion in well-established cell culture models of ovarian cancer cell lines (A2780 and SKOV-3). Furthermore, miR-450a overexpression resulted in higher rate of anoikis of the A2780 and SKOV-3 cells. In addition, as effort to identify miR-450a and miR-450b targets, we profiled the transcriptome of overexpressed miR-450a A2780 cells (A2780+pLVX+miR-450a) or miR-450b (A2780+pLVX+miR-450b) with control cell line (A2780+pLVX) using RNA-Seq. Among the most dysregulated pathways was the "Integrin family cell surface interactions" pathway. This finding is consistent with the migration and invasion phenotype we observed. While the RNA-Seq analysis revealed multiple dysregulated genes, the expression changes can be direct and indirect. The aim of this project is to find miR-450a and miR-450b targets to understand their function in ovarian cancer system better. We intend to perform Argonaute (AGO) PAR-CLIP (Photoactivatable Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) to comprehensively identify miRNA targets in SKOV3 and A2780 cells. PAR-CLIP is a next-generation sequencing based method which allows identifying RNA targets and binding sites of RNA-binding proteins at a transcriptome-wide scale and nucleotide resolution. AGO PAR-CLIP will reveal those transcripts under the direct control of miR-450a or miR-450b, as well as competition and synergy within the regulatory network controlled by miRNAs. PAR-CLIP method and analysis will be accomplished in collaboration with Dr. Markus Hafner, who developed it. This knowledge will allow us to pinpoint therapeutically valuable targets for treatment, diagnosis, and prognosis of ovarian cancer.

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Scientific publications
(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)
MUYS, BRUNA RODRIGUES; SOUSA, JOSANE F.; PLACA, JESSICA RODRIGUES; DE ARAUJO, LUIZA FERREIRA; SARSHAD, AISHE A.; ANASTASAKIS, DIMITRIOS G.; WANG, XIANTAO; LI, XIAO LING; DE MOLFETTA, GREICE ANDREOTTI; RAMAO, ANELISA; et al. miR-450a Acts as a Tumor Suppressor in Ovarian Cancer by Regulating Energy Metabolism. Cancer Research, v. 79, n. 13, p. 3294-3305, . (16/22307-9, 13/08135-2, 09/53853-5, 13/25119-0, 13/25326-6)

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