Clear 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 (TCGA) database were integrated to identify candidate miRs 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 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 EMT/MET 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.
Translational research : the journal of laboratory and clinical medicine. 2022 Jun 10 [Epub ahead of print]
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, 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, 4200-072, Porto, Portugal; Master in Molecular Medicine and Oncology, Faculty of Medicine-University of Porto (FMUP), Alameda Prof. Hernâni Monteiro, 4200-319, Porto, Portugal.. Electronic address: ., 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, 4200-072, Porto, Portugal. Electronic address: ., 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, 4200-072, Porto, Portugal. Electronic address: ., 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, 4200-072, Porto, Portugal; Department of Pathology, Portuguese Oncology Institute of Porto, Porto, Portugal. Electronic address: ., Department of Pathology, Portuguese Oncology Institute of Porto, Porto, Portugal. Electronic address: ., Department of Urology, Portuguese Oncology Institute of Porto (IPO Porto), Rua Dr. António Bernardino de Almeida, 4200-072, Porto, Portugal. Electronic address: ., Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Campus de Gualtar, Braga, Portugal; ICVS/3B's - PT Government Associate Laboratory, Braga/Guimarães, University of Minho, Campus de Gualtar, Braga, Portugal. Electronic address: ., Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Campus de Gualtar, Braga, Portugal; ICVS/3B's - PT Government Associate Laboratory, Braga/Guimarães, University of Minho, Campus de Gualtar, Braga, Portugal. Electronic address: ., Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Campus de Gualtar, Braga, Portugal; ICVS/3B's - PT Government Associate Laboratory, Braga/Guimarães, University of Minho, Campus de Gualtar, Braga, Portugal. Electronic address: ., 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, 4200-072, Porto, Portugal; 7Department of Pathology and Molecular Immunology, School of Medicine & Biomedical Sciences - University of Porto (ICBAS-UP), Rua de Jorge Viterbo Ferreira n.° 228, 4050-313, Porto, Portugal. Electronic address: ., 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, 4200-072, Porto, Portugal; Department of Pathology, Portuguese Oncology Institute of Porto, Porto, Portugal; 7Department of Pathology and Molecular Immunology, School of Medicine & Biomedical Sciences - University of Porto (ICBAS-UP), Rua de Jorge Viterbo Ferreira n.° 228, 4050-313, Porto, Portugal. Electronic address: ., 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, 4200-072, Porto, Portugal; 7Department of Pathology and Molecular Immunology, School of Medicine & Biomedical Sciences - University of Porto (ICBAS-UP), Rua de Jorge Viterbo Ferreira n.° 228, 4050-313, Porto, Portugal. Electronic address: .