MicroRNA Expression Profiling in Clear Cell Renal Cell Carcinoma: Identification and Functional Validation of Key miRNAs

<div><p>Objective</p><p>This study aims to profile dysregulated microRNA (miRNA) expression in clear cell renal cell carcinoma (ccRCC) and to identify key regulatory miRNAs in ccRCC.</p><p>Methods and Results</p><p>miRNA expression profiles in nine pairs of ccRCC tumor samples at three different stages and the adjacent, non-tumorous tissues were investigated using miRNA arrays. Eleven miRNAs were identified to be commonly dysregulated, including three up-regulated (miR-487a, miR-491-3p and miR-452) and eight down-regulated (miR-125b, miR-142-3p, miR-199a-5p, miR-22, miR-299-3p, miR-29a, miR-429, and miR-532-5p) in tumor tissues as compared with adjacent normal tissues. The 11 miRNAs and their predicted target genes were analyzed by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, and three key miRNAs (miR-199a-5p, miR-22 and miR-429) were identified by microRNA-gene network analysis. Dysregulation of the three key miRNAs were further validated in another cohort of 15 ccRCC samples, and the human kidney carcinoma cell line 786-O, as compared with five normal kidney samples. Further investigation showed that over-expression of miR-199a-5p significantly inhibited the invasion ability of 786-O cells. Luciferase reporter assays indicated that miR-199a-5p regulated expression of TGFBR1 and JunB by directly interacting with their 3’ untranslated regions. Transfection of miR-199a-5p successfully suppressed expression of TGFBR1 and JunB in the human embryonic kidney 293T cells, further confirming the direct regulation of miR-199a-5p on these two genes.</p><p>Conclusions</p><p>This study identified 11 commonly dysregulated miRNAs in ccRCC, three of which (miR-199a-5p, miR-22 and miR-429) may represent key miRNAs involved in the pathogenesis of ccRCC. Further studies suggested that miR-199a-5p plays an important role in inhibition of cell invasion of ccRCC cells by suppressing expression of TGFBR1 and JunB.</p></div

MiRNA expression profiling in ccRCC using microarray analysis.

<p>Unsupervised clustering of differentially expressed miRNAs in the three stages of ccRCC tumors (GI, GII and GIII) as compared with adjacent normal tissues. Three ccRCC tumor tissues from each stage, as well as the adjacent nontumorous tissues were randomly selected and subjected to global miRNA expression profiling. Differential miRNA expression was analyzed by comparison of the tumor tissues with adjacent normal tissues. The miRNAs with more than two-fold change in expression were considered to be differentially expressed. The expression of these miRNA candidates is illustrated in the heat map. The brightest green, black, and brightest red colors represent low, medium, and high expression of miRNAs, respectively.</p

Three key miRNAs and their predicted target genes in cancer-related pathways.

<p>Three key miRNAs and their predicted target genes in cancer-related pathways.</p

miR-199a-5p decreased invasive ability of ccRCC cells.

<p><b>(A)</b> Overexpression of miR-199a-5p in 786-O cells after miR-199a-5p mimics transfection compared with that in NC cells. <b>(B)</b> miR-199a-5p suppressed the invasive ability of 786-O cells as indicated by the transwell assay. Representative images from transwell assay (upper panel) and quantitative comparison of the invasive ability of cells transfected with miR-199a-5p mimics and NC mimics (lower panel) are shown.</p

miR-199a-5p directly suppressed expression of TGFBR1 and JunB in ccRCC.

<p><b>(A)</b> Relative luciferase activities in 293T cells co-transfected with psiCHECK luciferase reporter containing the miR-199a-5p recognition region and hsa-mir-199a-5p mimics or NC mimics(15 nM). <b>(B)</b> Relative luciferase activities in 293T cells co-transfected with psiCHECK luciferase reporter containing mutated miR-199a-5p recognition region in JunB (JunB-mut) or TGFBR1 (TGFBR-mut) and hsa-mir-199a-5p mimics or NC mimics (50 nM). Level of activity was calculated by normalizing <i>Renilla</i> luciferase to <i>Firefly</i> luciferase. P-values were determined by student <i>t</i>-test. Mean and SD were calculated from three independent experiments. <b>(C)</b> Western blotting showed the protein levels of TGFBR1 and JunB upon expression of miR-199a-5p mimics in human kidney carcinoma 786-O cells. β-actin was used as a loading control. Relative JunB and TGFBR1 protein levels were quantified and expressed as the ratio of JunB or TGFBR1 and β-actin.</p

MicroRNA-gene network in ccRCC.

<p><b>(A)</b> The top 10 Gene Ontology terms significantly enriched by predicted target genes of the up-regulated (A1) and down-regulated (A2) miRNAs in ccRCC. <b>(B)</b> The top 10 KEGG pathways significantly enriched by predicted target genes of the up-regulated (B1) and down-reguated (B2) miRNAs in ccRCC. <b>(C)</b> Interactive miRNA-gene networks between up-regulated (C1) or down-regulated (C2) miRNAs and their target genes. The red nodes represent the regulators (miRNAs), the grey nodes represent the targets (genes), and the green edges indicate direct interaction.</p