Epithelial-mesenchymal transition-associated microRNA/mRNA signature is linked to metastasis and prognosis in clear-cell renal cell carcinoma

Abstract

Clear-cell renal cell carcinomas (ccRCCs) are genetically heterogeneous tumors presenting diverse clinical courses. Epithelial-mesenchymal transition (EMT) is a crucial process involved in initiation of metastatic cascade. The aim of our study was to identify an integrated miRNA/mRNA signature associated with metastasis and prognosis in ccRCC through targeted approach based on analysis of miRNAs/mRNAs associated with EMT. A cohort of 230 ccRCC was included in our study and further divided into discovery, training and validation cohorts. EMT markers were evaluated in ccRCC tumor samples, which were grouped accordingly to EMT status. By use of large-scale miRNA/mRNA expression profiling, we identified miRNA/mRNA with significantly different expression in EMT-positive tumors and selected 41 miRNAs/mRNAs for training phase of the study to evaluate their diagnostic and prognostic potential. Fifteen miRNAs/mRNAs were analyzed in the validation phase, where all evaluated miRNA/mRNA candidates were confirmed to be significantly deregulated in tumor tissue. Some of them significantly differed in metastatic tumors, correlated with clinical stage, with Fuhrman grade and with overall survival. Further, we established an EMT-based stage-independent prognostic scoring system enabling identification of ccRCC patients at high-risk of cancer-related death. Finally, we confirmed involvement of miR-429 in EMT regulation in RCC cells in vitro.

Figure 1. A flow chart of the study design.

RP (renal parenchyma), RCC (renal cell carcinoma).

Figure 2. Expression levels of the EMT-associated miRNAs/mRNAs in renal parenchyma, tumors (all TNM stages), non-metastatic tumors (stage I + II), metastatic tumors (stage III + IV) and metastasis (validation phase of the study).

All comparisons are between two groups by use of Mann-Whitney test. RP (renal parenchyma), RCC (renal cell carcinoma), st.I + II (TNM stage I + II), st.III + IV (TNM stage III + IV), MET (metastasis).

Figure 3. EMT-associated miRNAs/mRNAs in relationship to the overall survival of ccRCC patients in validation phase of the study (cut-off values for prognostic stratification were adapted from training phase of the study).

Figure 4. Integrated EMT-based miRNAs/mRNAs prognostic model.

The best model for the prediction of 5-year overall survival consisted of 5 miRNAs (miR-200a, miR-200b, miR-200c, miR-429, miR-30a-3p) and 3 genes (C3orf52, CDH1, PAPSS2) (A,B). Independent validation of the integrated miRNA/mRNA EMT-signature by use of The Cancer Genome Atlas dataset KIRC (C). The predicted survival was assessed for the model without EMT-based miRNA/mRNA signature (D) and the model based only on the EMT-based miRNA/mRNA signature (age, gender, stage were fixed values) (E).

Figure 5. MiR-429 restores TGF-β-induced CDH1 suppression in renal cell carcinoma cell lines.

(* means p < 0.05).