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Functional analysis of 14 RNA binding proteins (RBPs) in two bladder urothelial carcinoma cell lines

Grant number: 16/05556-5
Support type:Scholarships abroad - Research Internship - Doctorate
Effective date (Start): July 01, 2016
Effective date (End): June 30, 2017
Field of knowledge:Biological Sciences - Genetics
Principal Investigator:Daisy Maria Favero Salvadori
Grantee:André Luiz Ventura Sávio
Supervisor abroad: Luiz Penalva
Home Institution: Faculdade de Medicina (FMB). Universidade Estadual Paulista (UNESP). Campus de Botucatu. Botucatu , SP, Brazil
Local de pesquisa : University of Texas Health Science Center at San Antonio (UTHSCSA), United States  
Associated to the scholarship:13/23279-0 - Bladder transitional cell carcinoma: a study of pos-transcriptional modifications and RNA binding proteins, BP.DR

Abstract

Bladder cancer has a high cost to health system, especially because of routine clinical monitoring (urine cytology and cystoscopy) and high recurrence rates. Due to the complexity of biological systems, the knowledge about the molecular mechanisms responsible for urothelial carcinomas is scarce. Literature shows that there is involvement of a complex network of pathways linking a large number of cellular processes, such as apoptosis, proliferation, invasion, growth, etc. In the first part of this study, we identified 14 RNA-binding proteins (RPBs are post-transcriptional regulators of gene expression involved in splicing, polyadenylation, stability, degradation and transport of RNAs and translation), which are related with poor prognosis for the bladder cancer. Now, the objective is to carry out the functional analysis of these 14 RBPs (NOCT, CELF2, ENDOU, EXO1, EZH2, IFIT2, MOV10L1, MSI, PEG10, PTRF, TERT, TRIM71, WARS and YBX2). For this purpose, two bladder urothelial carcinoma cell lines will be used: i) UM-UC-3, derived from an invasive tumor and with high in vitro invasion and migration, ii) T24, from an invasive tumor with the TP53 allele encoding an in-frame deletion of tyrosine 126. The small interference RNA (siRNA) assay followed by cell viability, cell proliferation, caspase 3/7 activity evaluation, knockdown quantification by real-time PCR and RNA-seq and CLIP from the two RBPs with good results in the preliminary functional assay in vitro, will be performed. The RNAseq aimed identified the changes in mRNA levels and splicing triggered by RPB target, and CLIP assay can helps to understand the pathway of each RBPs and establish its relationship with tumorigenesis. We expect the results may contribute for better understanding the tumorigenesis mechanisms of bladder cancer, and also provide relevant information for early prognosis biomarkers identification and for treatment of urothelial carcinomas.