miRNA-target network reveals miR-124as a key miRNA contributing to clear cell renal cell carcinoma aggressive behaviour by targeting CAV1 and FLOT1

Abstract

Clear cell renal cell carcinoma (ccRCC) is an aggressive tumor with frequent metastatic rate and poor survival. Integrated analyses allow understanding the interplay between different levels of molecular alterations.We integrated miRNA and gene expression data from 458 ccRCC and 254 normal kidney specimens to construct a miRNA-target interaction network.We identified the downregulated miR-124-3p, -30a-5p and -200c-3p as the most influential miRNAs in RCC pathogenesis.miR-124-3p and miR-200c-3p expression showed association with patient survival, miR-30a-5p was downregulated in metastases compared to primary tumors. We used an independent set of 87 matched samples for validation. We confirmed the functional impact of these miRNAs by in vitro assays. Restoration of these miRNAs reduced migration, invasion and proliferation. miR-124-3p decreased the S phase of cell cycle, as well. We compared transcriptome profiling before and after miRNA overexpression, and validated CAV1 and FLOT1 as miR-124-3p targets. Patients with higher CAV1 and FLOT1 had lower miR-124-3p expression and shorter overall survival.We hypothesize that these three miRNAs are fundamental contributing to ccRCC aggressive/metastatic behavior; and miR-124-3p especially has a key role through regulating CAV1 and FLOT1 expression. Restoration of the levels of these miRNAs could be considered as a potential therapeutic strategy for ccRCC.

Keywords: CAV1; FLOT1; integrated analysis; miR-124-3p; renal cell carcinoma.

Figure 1. miRNA-target network of ccRCC

Organic plot of miRNA-target network of ccRCC. Node's colour and size represents the number of interaction (node degree); edge's colour and size indicates the significance of interactions (edge betweeness).

Figure 2. miR-124-3p, -30a-5p and -200c-3p expression in ccRCC and miRNA expression association with patient survival

A: miR-124-3p, -30a-5p and -200c-3p are downregulated in ccRCC with -2.46, -1.41 and -9.39 fold change, respectively (p < 0.001) compared to normal kidney. N_Normal: 66, N_ccRCC: 499 – data were extracted from TCGA dataset. B: Survival analysis performed on our independent patient set of 62 ccRCCs. Patients having lower miR-124-3p or higher miR-200c-3p expression have worse disease-free and overall survival. There was no significant association between miR-30a-5p expression and survival. C: miRNA expression in primary ccRCC and matched metastatic specimens. miR-124 and -30a-5p were downregulated with 4.16 and 5.98 folds (p = 0.09 and p = 0.046). miR-200c-3p was overexpressed in metastases compared to the primary tumors (105 fold change; p < 0.0001). Columns and bars represent mean±SD, Stars (*) indicate statistical significance (p < 0.05).

Figure 3. Gene Ontology (GO) analysis of ccRCC upon the effect of miR-124-3p, -30a-5p and -200c-3p

A: Functional gene ontology clustering of ccRCC miRNA-target network members. B: Functional gene ontology analysis of miR-124, miR-30a-5p and miR-200c-3p targets evaluated by transcriptome profiling following triple-miRNAtransfection in 786-O and Caki-2 cells. Circles represent GO enriched categories. Colours and axis Y indicate log10(Benjamini-Hochberg FDR q-values).

Figure 4. miR-124-3p, -30a-5p and -200c-3p effect on migration and invasion of kidney cancer cell lines

A: miRNA effect on transwell migration and invasion of 786-O and Caki-2 cells. miR-124-3p, -30a-5p and 200c-3p overexpression decreased 786-O cells migration by 40%, 39% and 47%, respectively (p < 0.001), and reduced Caki-2 cell migration by 68%, 28% and 66%, respectively (p < 0.001). miR-124-3p and miR-200c-3p decreased cell invasion by 55% and 61%, respectively (p < 0.001) in 786-O cells and by 74% and 77% (p < 0.001) in Caki-2 cells. miR-30a-5p also inhibited invasion of 786-O cells by 35% (p < 0.001) but had no significant effect on Caki-2 cells (p = 0.07). Columns and bars represent mean±SEM. B: Representative photomicrographs showing the effect of miR-124-3p overexpression on the migration rate of the 786-O RCC cell line. The top row shows the cells at the time of wounding (0 h), and the bottom row shows cellular migration after 6 hours. Overexpression of miR-124-3p significantly decreased the rate of cell migration, with incomplete wound closure after 6h, compared to controls. Representative bar graph showing the effect of miR-124-3p on cellular migration.

Figure 5. miR-124-3p, -30a-5p and -200c-3p effect proliferation and cell cycle

A: Triple-miRNA transfection decreased cell proliferation/viability by 29.91%, 27.05% and 26.64% in 786-O, Caki-2 and ACHN cells using WST-1 assay. Individual miR-124-3p transfection also inhibited proliferation/viability of Caki-2 and ACHN cells by 18.12% and 24.75%. Columns and bars represent mean±SD, B: miR-124-3p mimic effect on cell cycle assessed by flow cytometry analysis. Representative pictures of cell cycle phase distribution after transfection with either Non-targeting Control or miR-124-3p mimic in different kidney cancer cell lines. C: normalized average dG1G0, dS and dG2M distribution (N = 3). D: miR-124-3p effect on proliferation investigated by CFSE flow cytometry analysis. Representative normalized CFSE dilution after transfection with averaged proliferation index, PI (N = 3). Data are presented as means of average percentage of cells ± SD. Stars (*) indicate statistical significance (p < 0.05), ns: non-significant.

Figure 6. CAV1 and FLOT1 protein expression and association with survival in clinical specimens and validation as miR-124-3p targets

miR-124-3p targets expression in ccRCC. Scatter plots show CAV1 (A) and FLOT1 (B) protein expression in 23 matched ccRCC vs. normal kidney specimens with representative western blot images. CAV1 and FLOT1 were upregulated with 21 and 1.5 folds, respectively (p < 0.01 and p = 0.03) in tumour tissues compared to their matched normals. C: miR-124-3p transfection resulted in CAV1 mRNA downregulation with 3.9, 5.8 and 5.11 folds (p < 0.001) in 786-O, ACHN and Caki-2 cells, respectively. FLOT1 expression was also reduced but this did not reach statistical significance. D-E: Both CAV1 and FLOT1 protein were downregulated following miRNA transfection by 52%, 55%, 66% (p < 0.001) and 39%, 48%, 44% (p < 0.05) in 786-O, ACHN and Caki-2 cells, respectively. F-G: Kaplan-Meier analysis showing that higher CAV1 or FLOT1 expressions are associated with worse survival of ccRCC patients. Columns and bars represent mean±SD, stars (*) indicate statistical significance (p < 0.05).