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Epigenetically-regulated miR-30a/c-5p directly target TWF1 and hamper ccRCC cell aggressiveness

  • GONÇALO OUTEIRO-PINHO
    Affiliations
    Cancer Biology and Epigenetics Group, Research Center of IPO Porto (CI-IPOP) / [email protected] (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
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  • DANIELA BARROS-SILVA
    Affiliations
    Cancer Biology and Epigenetics Group, Research Center of IPO Porto (CI-IPOP) / [email protected] (Health Research Network), Portuguese Oncology Institute of Porto (IPO Porto) / Porto Comprehensive Cancer Center (Porto.CCC), R. Dr. António Bernardino de Almeida, Porto, Portugal
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  • FILIPA MOREIRA-SILVA
    Affiliations
    Cancer Biology and Epigenetics Group, Research Center of IPO Porto (CI-IPOP) / [email protected] (Health Research Network), Portuguese Oncology Institute of Porto (IPO Porto) / Porto Comprehensive Cancer Center (Porto.CCC), R. Dr. António Bernardino de Almeida, Porto, Portugal
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  • JOÃO LOBO
    Affiliations
    Cancer Biology and Epigenetics Group, Research Center of IPO Porto (CI-IPOP) / [email protected] (Health Research Network), Portuguese Oncology Institute of Porto (IPO Porto) / Porto Comprehensive Cancer Center (Porto.CCC), R. Dr. António Bernardino de Almeida, Porto, Portugal

    Department of Pathology, Portuguese Oncology Institute of Porto, Porto, Portugal
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  • ISA CARNEIRO
    Affiliations
    Department of Pathology, Portuguese Oncology Institute of Porto, Porto, Portugal
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  • ANTÓNIO MORAIS
    Affiliations
    Department of Urology, Portuguese Oncology Institute of Porto (IPO Porto), Rua Dr. António Bernardino de Almeida, Porto, Portugal
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  • EDUARDA P. MARTINS
    Affiliations
    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
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  • CÉLINE S. GONÇALVES
    Affiliations
    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
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  • BRUNO M. COSTA
    Affiliations
    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
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  • MARGARETA P. CORREIA
    Affiliations
    Cancer Biology and Epigenetics Group, Research Center of IPO Porto (CI-IPOP) / [email protected] (Health Research Network), Portuguese Oncology Institute of Porto (IPO Porto) / Porto Comprehensive Cancer Center (Porto.CCC), R. Dr. António Bernardino de Almeida, Porto, Portugal

    Department of Pathology and Molecular Immunology, School of Medicine & Biomedical Sciences - University of Porto (ICBAS-UP), Porto, Portugal
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  • RUI HENRIQUE
    Affiliations
    Cancer Biology and Epigenetics Group, Research Center of IPO Porto (CI-IPOP) / [email protected] (Health Research Network), Portuguese Oncology Institute of Porto (IPO Porto) / Porto Comprehensive Cancer Center (Porto.CCC), R. Dr. António Bernardino de Almeida, Porto, Portugal

    Department of Pathology, Portuguese Oncology Institute of Porto, Porto, Portugal

    Department of Pathology and Molecular Immunology, School of Medicine & Biomedical Sciences - University of Porto (ICBAS-UP), Porto, Portugal
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  • CARMEN JERÓNIMO
    Correspondence
    Reprint requests: Carmen Jerónimo, Cancer Biology and Epigenetics Group, Research Center of IPO Porto (CI-IPOP) / [email protected] (Health Research Network), Portuguese Oncology Institute of Porto (IPO Porto) / Porto Comprehensive Cancer Center (Porto.CCC), R. Dr. António Bernardino de Almeida, Blg F, 1st floor, 4200-072 Porto, Portugal.
    Affiliations
    Cancer Biology and Epigenetics Group, Research Center of IPO Porto (CI-IPOP) / [email protected] (Health Research Network), Portuguese Oncology Institute of Porto (IPO Porto) / Porto Comprehensive Cancer Center (Porto.CCC), R. Dr. António Bernardino de Almeida, Porto, Portugal

    Department of Pathology and Molecular Immunology, School of Medicine & Biomedical Sciences - University of Porto (ICBAS-UP), Porto, Portugal
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      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 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.

      Abbreviations:

      ccRCC (clear cell renal cell carcinoma), chRCC (chromophobe renal cell carcinoma), DAC (5-aza-2-deoxycytidine decitabine), DFS (disease-free survival), DMSO (dimethyl sulfoxide), dSarc (ccRCC tumors with sarcomatoid differentiation), DSS (disease-specific survival), EMT (epithelial-mesenchymal transition), FFPE (formalin-fixed paraffin-embedded), GDC (Genomic Data Commons), HR (hazard ratio), IHC (immunohistochemistry), lncRNA (long non-coding RNA), MET (mesenchymal-epithelial transition), MFS (metastasis-free survival), miR (microRNA), miR-30a/c-5p (miR-30a-5p and miR-30c-5p), miR-30c-5pme (miR-30c-5p promoter methylation levels), MRE (miRNA response elements), MTX (metastatic tissue), NC (negative control), OD (optical density), ON (overnight), OS (overall survival), PCT (probability of conserved targeting), PIC (proteases inhibitor cocktail), pRCC (papillary renal cell carcinoma), PT (primary tumors of patients without disease progression), PT-MTX (primary tumors of patients which experienced subsequent metastatic dissemination), RCC (renal cell carcinoma), RFS (recurrence-free survival), RIPA (radioimmunoprecipitation assay), RNAi (RNA interference), RNT (renal normal tissue), SDS (sodium dodecyl sulfate), SDS-PAGE (SDS polyacrylamide gel electrophoresis), TCGA (The Cancer Genome Atlas)
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