Fig 1.
Interaction Network between 3 differentially expressed miRNAs (downregulated M1 vs M0, green) and differentially expressed mRNAs (upregulated M1 vs M0, red) from ccRCC patients.
Fig 2.
Expression of miR-146a-5p in ccRCC tissue samples.
RT-qPCR analysis of the expression of miR-146a-5p in ccRCC tissue samples was performed. The data are represented in a scatter dot plots with means for normal, non-malignant renal tissue samples (N; n = 10), primary tumor samples without diagnosed metastasis (ccRCC-M0; n = 10), and primary tumor samples with diagnosed metastasis (ccRCC-M1; n = 10). Normalization was assessed with the reference miRNA combination miR-28, miR-103, and miR-106a [18], (S2 Fig). Statistical differences were calculated using the Mann-Whitney U test between the groups.
Table 1.
Expression of selected miRNAs in non-malignant, ccRCC-M0 and ccRCC-M1 samples.
Fig 3.
Expression of putative mRNAs targeted of miR-146a-5p.
The relative mRNA expression levels of the potential miR-146a-5p targets BRCA1, MCM10, CDKN3, CXCL8/IL8, and UHRF1 were measured in duplicates in a pool of normal renal tissue and tissue samples from primary ccRCC-M0 and ccRCC-M1 patients by RT-qPCR. Data were normalized with PPIA reference gene [19], (S2 Fig). BRCA1, CDKN3, MCM10, CXCL8/IL8, and UHRF1 are lower in tissue samples of ccRCC without metastasis compared to ccRCC with metastasis.
Fig 4.
Effect of miR-146a-5p on putative mRNA target levels in 786-O and ACHN cells.
Application of miR-146a-5p mimic and miR-146a-5p inhibitor effects mRNA target expression in cell lines. Data were normalized with PPIA reference gene [19]. NC1 = negative control.
Fig 5.
Effect of miR-146a-5p on CXCL8 protein levels in 786-O and ACHN cells.
Application of miR-146a-5p mimic induced a significant reduction of CXCL8 protein expression in (A) 786-O cells and (B) ACHN cells, whereas a miR-146a-5p inhibitor rescued the CXCL8 expression levels in both cell lines. NC1 = negative control.
Fig 6.
Effect of miR-146a-5p on UHRF1 protein levels in 786-O and ACHN cells.
(A) Western blots and (B, C) their densitometric analyses showed a reduced UHRF1 protein expression in 786-O and ACHN cells after application of the miR-146a-5p mimic, whereas the miR-146a-5p inhibitor had no effect on UHRF1 expression levels in both cell lines. NC1 = negative control.