Integrated microRNA and mRNA Signature Associated with the Transition from the Locally Confined to the Metastasized Clear Cell Renal Cell Carcinoma Exemplified by miR-146-5p

Abstract

BackgroundMicroRNAs (miRNAs) regulate gene expression by interfering translation or stability of target transcripts. This interplay between miRNA and their mRNA has been proposed as an important process in cancer development and progression. We have investigated molecular networks impacted by predicted mRNA targets of differentially expressed miRNAs in patients with clear cell renal cell carcinoma (ccRCC) diagnosed with or without metastasis.Material and MethodsmiRNA and mRNA microarray expression profiles derived from primary ccRCC from patients with (16 samples) or without diagnosed metastasis (22 samples) were used to identify anti-correlated miRNA-mRNA interaction in ccRCC. For this purpose, Ingenuity pathway analysis microRNA Target Filter, which enables prioritization of experimentally validated and predicted mRNA targets was used. By applying an expression pairing tool, the analysis was focused on targets exhibiting altered expression in our analysis, finding miRNAs and their target genes with opposite or same expression. The resulting identified interactions were revalidated by RT-qPCR in another cohort of ccRCC patients. A selection of the predicted miRNA-mRNA interactions was tested by functional analyses using miRNA knockdown and overexpression experiments in renal cancer cell lines.ResultsAmong the significantly differentially expressed miRNAs, we have identified three miRNAs (miR-146a-5p, miR-128a-3p, and miR-17-5p) that were upregulated in primary tumors from patients without metastasis and downregulated in primary tumors from patients with metastasis. We have further identified mRNA targets, which expression were inversely correlated to these 3 miRNAs, and have been previously experimentally demonstrated in cancer setting in humans. Specifically, we showed that CXCL8/IL8, UHRF1, MCM10, and CDKN3 were downregulated and targeted by miR-146a-5p. The interaction between miR-146a-5p and their targets CXCL8 and UHRF1 was validated in cell culture experiments.ConclusionsWe identified novel target genes of dysregulated miRNAs, which are involved in the transition from primary RCC without metastases into tumors generating distant metastasis.

Inhibiting WNT and NOTCH in renal cancer stem cells and the implications for human patients

“…GO term enrichment and Kegg pathway analyses were performed with DAVID 38 . The gene expression analyses of 28 fresh frozen ccRCC specimens has been performed previously at the Charité-University hospital and only the data were used for this study (GEO Accession Code: GSE66270 and GSE66271, for patient information see Supplementary Table 2) 41 . Expression of stem cell genes was analyzed using the GeneSpring GX software (Agilent), and supervised hierarchical clustering was performed in Genesis 69 .…”

“…To confirm that the stem cell signature was associated with the clinical course of ccRCC, we examined gene expression data that had previously been obtained from 28 additional ccRCC patients (Supplementary Table 2) 41 . In these analyses, we included all genes with a FC > 1.…”

UHRF1: The key regulator of epigenetics and molecular target for cancer therapeutics

“…84 In renal cell carcinoma also miR-146a-5p targeted UHRF1. 85 miR-193a-3p has been shown to downregulate UHRF1 in a highly metastatic lung cancer cell line (SPC-A-1sci) resulting in reduced metastasis. 86 Blocking of miR-9 expression with antagonistic oligonucleotide in HCT116 and HT29 colorectal cancer cells resulted in enhanced expression of UHRF1 (Figure 3).…”

Prognostic signatures for renal cancer as identified by long non-coding and miRNA competing endogenous network analysis.

“…Three RNA datasets (mRNA, lncRNA and miRNA) were selected in this study. In these datasets, GSE66270, GSE40914 and GSE71302 were analyzed as they were associated with renal cancer (30)(31)(32). GSE66270 (mRNA) of the Affymetrix Human Genome U133 Plus 2.0 Array (GPL570) platform consisted of 28 samples including 14 renal cancer and 14 normal samples.…”