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| Author(s): |
André Luiz Ventura Savio
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | Botucatu. 2019-03-21. |
| Institution: | Universidade Estadual Paulista (Unesp). Instituto de Biociências. Botucatu |
| Defense date: | 2019-01-31 |
| Advisor: | Daisy Maria Favero Salvadori |
| Abstract | |
Urothelial carcinoma represents one of the most common types of urinary neoplasms, with high rates of recurrence, aggressiveness and progression to invasive muscular disease. Due to the complexity of the biological systems, little is known about the molecular mechanisms responsible for urothelial carcinomas. In recent years, the increase of bioinformatics tools has enabled the identification of new molecules and molecular mechanisms involved in carcinogenesis. In this study, two approaches were conducted aiming to identify new potential biomarkers for low and high grades urothelial tumors. Initially, data from RNA sequencing showed the levels of gene expression and splicing profile for urothelial tumors (low and high grades) and normal bladder tissues obtained from the biorepository of the University of São Paulo Medical School (FMUSP), Brazil. The gene expression profiling demonstrated modulated expression in genes related to the TP53 pathway in both low and high grade tumors. In addition, the splicing data showed that the preferentially affected genes were those related to cell cycle, adhesion, migration and RNA processing. The high-grade tumors presented increased expression of genes related to chemotaxis (GREM1, S100A12, NR4A1, IL6, CCL20, CXCL8, S100A9, CXCL10, CXCL11 and CCL7) and neuronal functions (EPHB2, CNTNAP2, KCNQ3, TENM2, RDH12, DPF1, SHISA9, SLC30A3, MME and MSI1). Furthermore, splicing modification in transcriptional factors (GAS5, RPL10, RPL13A and RPL37A) with potential impact on protein production were also identified, but, exclusively, in high-grade tumors. In the second approach, a functional study was conducted aiming to identify the role of RNA binding proteins (RBPs) in urothelial carcinogenesis. For this, the expression genes related to 1,542 RBPs were measured from 405 cases of urothelial carcinoma obtained from the TCGA (384 high and 21 low grade tumors). A total of 236 RBPs with oncogenic potential were identified in high-grade tumors, being 14 (NOCT, CELF2, ENDOU, EXO1, EZH2, IFIT2, MOV10L1, MSI, PEG10, PTRF, TERT, TRIM71, WARS and YBX2) also related to worse prognosis and, therefore, selected for the functional 14 study. The RBPs expression validation showed that 8/14 (EXO, EZH2, NOCT, TERT, MOV10L1, MSI1, WARS and YBX2) were also highly expressed in the tumors samples obtained from FMUSP, Brazil. Functional analysis in three cell lines (UMUC3, T24, and J82) showed that the silencing of each RBPs encoding gene (total of 14) resulted in decreased cell proliferation and viability, and increased rates of apoptosis. The MSI1 (musashi-1 knockdown) demonstrated a strong impact on cell viability, proliferation, migration, invasion and apoptosis, and also resulted in decreased resistance to in vitro chemo and radiotherapies. Data from the RNA sequencing after MSI1 knockdown showed impact on the expression of genes related to epithelial cell differentiation, Wnt-pathway and chemotaxis. The musashi-1 target analysis (by crosslinking immunoprecipitation- CLIP) showed that this RBP preferentially bound to the 3'UTR and intronic regions of mRNA related to translation, RNA processing and Wnt, TP53, PDGF,and CCKR pathways. Taken together, data from the two approaches allowed the identification of new panel of candidate biomarkers, and a new therapeutic target (musashi1) for urothelial high-grade tumors. (AU) | |
| FAPESP's process: | 13/23279-0 - Bladder transitional cell carcinoma: a study of pos-transcriptional modifications and RNA binding proteins |
| Grantee: | André Luiz Ventura Sávio |
| Support Opportunities: | Scholarships in Brazil - Doctorate |