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

Butz H, Szabó PM, Khella HW, Nofech-Mozes R, Patocs A, Yousef GM - Oncotarget (2015)

Bottom Line: 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.Restoration of the levels of these miRNAs could be considered as a potential therapeutic strategy for ccRCC.

View Article: PubMed Central - PubMed

Affiliation: Department of Laboratory Medicine and The Keenan Research Centre for Biomedical Science of St. Michael's Hospital, Toronto, Canada.

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.

No MeSH data available.


Related in: MedlinePlus

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

Mentions: Because miR-124-3p accomplished only a slight effect on cell proliferation next we focused its influence on migration/invasion processes. To understand the mechanism by which miR-124-3p can affect tumor cell migration and invasion, we selected two targets of miR-124-3p involved in cell migration-invasion for further validation. Caveolin 1 (CAV1) was downregulated by microarray with −4.59 and −8.00 fold change (p < 0.001 and =0.007) following miR-124-3p transfection, and it was involved in pathways related to migration (Supplementary Table 5A). Flotillin 1 (FLOT1) is documented in the literature to be implicated in cell migration and was a validated target of miR-124-3p in breast cancer [13]. We investigated CAV1 and FLOT1 protein expression by Western blot in 23 pairs of normal-ccRCC specimens, and found CAV1 protein to be upregulated with an average 20.8 fold and FLOT1 with 1.47 fold in ccRCC compared to normal counterparts form the same patient (Figure 6A-6B). As miRNAs having a so-called fine tuning effect on the target expression 1,47 fold change on protein level can be resulted as a miRNA effect. In many cases miRNAs do not lead to expression change on mRNA level but only on protein level. To include these possibilities as well we selected FLOT1 as a potential second target beside CAV1. We further assessed the effect of miR-124-3p overexpression on these two targets at the mRNA level (by RT-qPCR) and protein level (by Western blot analysis) in 786-O, ACHN and Caki-2 cell lines. miR-124-3p overexpression resulted in a significant reduction of the expression of CAV1 at the mRNA level (Figure 6C) and the expression of CAV1 and FLOT1 at protein level (Figure 6D-6E).


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

Butz H, Szabó PM, Khella HW, Nofech-Mozes R, Patocs A, Yousef GM - Oncotarget (2015)

CAV1 and FLOT1 protein expression and association with survival in clinical specimens and validation as miR-124-3p targetsmiR-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).
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Figure 6: CAV1 and FLOT1 protein expression and association with survival in clinical specimens and validation as miR-124-3p targetsmiR-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).
Mentions: Because miR-124-3p accomplished only a slight effect on cell proliferation next we focused its influence on migration/invasion processes. To understand the mechanism by which miR-124-3p can affect tumor cell migration and invasion, we selected two targets of miR-124-3p involved in cell migration-invasion for further validation. Caveolin 1 (CAV1) was downregulated by microarray with −4.59 and −8.00 fold change (p < 0.001 and =0.007) following miR-124-3p transfection, and it was involved in pathways related to migration (Supplementary Table 5A). Flotillin 1 (FLOT1) is documented in the literature to be implicated in cell migration and was a validated target of miR-124-3p in breast cancer [13]. We investigated CAV1 and FLOT1 protein expression by Western blot in 23 pairs of normal-ccRCC specimens, and found CAV1 protein to be upregulated with an average 20.8 fold and FLOT1 with 1.47 fold in ccRCC compared to normal counterparts form the same patient (Figure 6A-6B). As miRNAs having a so-called fine tuning effect on the target expression 1,47 fold change on protein level can be resulted as a miRNA effect. In many cases miRNAs do not lead to expression change on mRNA level but only on protein level. To include these possibilities as well we selected FLOT1 as a potential second target beside CAV1. We further assessed the effect of miR-124-3p overexpression on these two targets at the mRNA level (by RT-qPCR) and protein level (by Western blot analysis) in 786-O, ACHN and Caki-2 cell lines. miR-124-3p overexpression resulted in a significant reduction of the expression of CAV1 at the mRNA level (Figure 6C) and the expression of CAV1 and FLOT1 at protein level (Figure 6D-6E).

Bottom Line: 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.Restoration of the levels of these miRNAs could be considered as a potential therapeutic strategy for ccRCC.

View Article: PubMed Central - PubMed

Affiliation: Department of Laboratory Medicine and The Keenan Research Centre for Biomedical Science of St. Michael's Hospital, Toronto, Canada.

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.

No MeSH data available.


Related in: MedlinePlus