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)To read this article in full you will need to make a payment
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Article info
Publication history
Published online: June 10, 2022
Accepted:
June 6,
2022
Received in revised form:
May 9,
2022
Received:
December 21,
2021
Identification
Copyright
© 2022 Elsevier Inc. All rights reserved.