• Gonçalo Outeiro-Pinho, Daniela Barros-Silva, Filipa Moreira-Silva, João Lobo, Isa Carneiro, António Morais, Eduarda P Martins, Céline S Gonçalves, Bruno M Costa, Margareta P Correia, Rui Henrique, and Carmen Jerónimo.
• Cancer Biology and Epigenetics Group, Research Center of IPO Porto (CI-IPOP) / RISE@CI-IPOP (Health Research Network), Portuguese Oncology Institute of Porto (IPO Porto) / Porto Comprehensive Cancer Center (Porto.CCC), R. Dr. António Bernardino de Almeida, Porto, Portugal; Master in Molecular Medicine and Oncology, Faculty of Medicine-University of Porto (FMUP), Alameda Prof. Hernâni Monteiro, Porto, Portugal.
• Transl Res. 2022 Nov 1; 249: 110127110-127.

AbstractClear cell renal cell carcinoma (ccRCC) is highly prone to metastasize and displays an extremely low 5-year survival rate. Not only miRNAs (miRs) are key gene expression regulators but can also be epigenetically modified. Abnormal miR expression has been linked with epithelial-mesenchymal transition (EMT)-driven ccRCC progression. MiR-30a/c-5p were found downregulated in ccRCC and associated with aggressiveness. Herein, we sought to unravel miR-30a/c-5p mechanistic role in ccRCC. RNA sequencing and genome-wide methylome data of ccRCC and normal tissue samples from The Cancer Genome Atlas database were integrated to identify candidate miRs cytosine-phosphate-guanine (CpG) loci deregulated in ccRCC. TargetScan was searched to identify miR putative targets. MiR-30a/c-5p expression and promoter methylation was evaluated in vitro, by PCR. Western blot, functional and luciferase assays were performed after cell transfection with either pre-miR, antimiR, or siRNA against twinfilin-1 (TWF1). Immunohistochemistry (IHC) was performed in ccRCC tissues. We found miR-30c-5p downregulation and aberrant promoter methylation in ccRCC tissues. In vitro studies revealed concomitant miR-30a/c-5p downregulation and increased promoter methylation, as well as a significant re-expression following decitabine treatment. Functional assays demonstrated that both miRs significantly decreased cell aggressiveness and the protein levels of EMT-promoting players, while upregulating epithelial markers, namely Claudin-1 and ZO-1. Importantly, we confirmed TWF1 as a direct target of both miRs, and its potential involvement in epithelial-mesenchymal transition/mesenchymal-epithelial transition regulation. IHC analysis revealed higher TWF1 expression in primary tissues from patients that developed metastases, after surgical treatment. Our results implicate miR-30a/c-5p in ccRCC cells' aggressiveness attenuation by directly targeting TWF1 and hampering EMT.Copyright © 2022 Elsevier Inc. All rights reserved.

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