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MiR-192 directly binds and regulates Dicer1 expression in neuroblastoma.

Feinberg-Gorenshtein G, Guedj A, Shichrur K, Jeison M, Luria D, Kodman Y, Ash S, Feinmesser M, Edry L, Shomron N, Weizman A, Yaniv I, Avigad S - PLoS ONE (2013)

Bottom Line: We were able to show through a dual luciferase assay and side-directed mutational analysis that miR-192 directly binds the 3' UTR of Dicer1 on positions 1232-1238 and 2282-2288.An increase in cell viability, proliferation and migration rates were evident in NB cells transfected with miR-192-mimic.Yet, there was a significant decrease in proliferation when NB cells were transfected with an miR-192-inhibitor We suggest that miR-192 might be a key player in NB by regulating Dicer1 expression.

View Article: PubMed Central - PubMed

Affiliation: Molecular Oncology, Felsenstein Medical Research Center, Petah Tikva, Israel ; Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.

ABSTRACT
Neuroblastoma (NB) arises from the embryonic neural crest and is the most common extracranial solid tumor in children under 5 years of age. Reduced expression of Dicer1 has recently been shown to be in correlation with poor prognosis in NB patients. This study aimed to investigate the mechanisms that could lead to the down-regulation of Dicer1 in neuroblastoma. We used computational prediction to identify potential miRs down-regulating Dicer1 in neuroblastoma. One of the miRs that were predicted to target Dicer1 was miR-192. We measured the levels of miR-192 in 43 primary tumors using real time PCR. Following the silencing of miR-192, the levels of dicer1 cell viability, cell proliferation and migration capability were analyzed. Multivariate analysis identified miR-192 as an independent prognostic marker for relapse in neuroblastoma patients (p=0.04). We were able to show through a dual luciferase assay and side-directed mutational analysis that miR-192 directly binds the 3' UTR of Dicer1 on positions 1232-1238 and 2282-2288. An increase in cell viability, proliferation and migration rates were evident in NB cells transfected with miR-192-mimic. Yet, there was a significant decrease in proliferation when NB cells were transfected with an miR-192-inhibitor We suggest that miR-192 might be a key player in NB by regulating Dicer1 expression.

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3′UTR of Dicer 1 is directly targeted by miR-192.The dual luciferase assay detected that Dicer1 is modulated by miR-192 in NB cell lines. The relative luciferase unit (RLU) was measured in SHEP (A) or NUB (B) cells.A. The dual-luciferase assay resulted in a significant reduction of RLU of WT Dicer1 (3' UTR of Dicer1 wild type) following transfection with miR-192-vec (*p=0.049).B. Following transfection with miR-192 mimic, WT Dicer1 RLU was significantly decreased (*p=0.0003). Following mutagenesis, cells were transfected with Dicer1 plasmid in which mutations were introduced in all three BSs of Dicer1 (MUT ALL); active BS1 (mutated at BS2+BS3)(* p=0.004); active BS2 (mutated at BS1+BS3) (* p=0.04) and active BS3 (mutated at BS1+BS2). Values are expressed as the mean ± SE of combined results from three independent experiments.
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pone-0078713-g006: 3′UTR of Dicer 1 is directly targeted by miR-192.The dual luciferase assay detected that Dicer1 is modulated by miR-192 in NB cell lines. The relative luciferase unit (RLU) was measured in SHEP (A) or NUB (B) cells.A. The dual-luciferase assay resulted in a significant reduction of RLU of WT Dicer1 (3' UTR of Dicer1 wild type) following transfection with miR-192-vec (*p=0.049).B. Following transfection with miR-192 mimic, WT Dicer1 RLU was significantly decreased (*p=0.0003). Following mutagenesis, cells were transfected with Dicer1 plasmid in which mutations were introduced in all three BSs of Dicer1 (MUT ALL); active BS1 (mutated at BS2+BS3)(* p=0.004); active BS2 (mutated at BS1+BS3) (* p=0.04) and active BS3 (mutated at BS1+BS2). Values are expressed as the mean ± SE of combined results from three independent experiments.

Mentions: To validate the relative relevance of each one of the potential BSs of miR-192 in the 3′ untranslated region (3′UTR) of Dicer1, two different dual-luciferase reporter assays were carried out in NB cell lines. The dual-luciferase assay resulted in a significant reduction of 30% in the relative luciferase unit (RLU) of Dicer1 as was evident in SHEP cell line (non-MYCNA) transfected with miR-192-vec (p=0.049; Figure 6A). Moreover, a significant decrease of 59% in the level of Dicer1 RLU was evident in the NUB6 cell line (MYCNA) after transfection with the miR-192 mimic (p=0.0003; Figure 6B). Following transfection with miR-192 inhibitor the Dicer1 RLU returned to the control basal level (determined by transfection of scrambled miR sequences). These results suggest that miR-192 expression regulates Dicer1 protein expression.


MiR-192 directly binds and regulates Dicer1 expression in neuroblastoma.

Feinberg-Gorenshtein G, Guedj A, Shichrur K, Jeison M, Luria D, Kodman Y, Ash S, Feinmesser M, Edry L, Shomron N, Weizman A, Yaniv I, Avigad S - PLoS ONE (2013)

3′UTR of Dicer 1 is directly targeted by miR-192.The dual luciferase assay detected that Dicer1 is modulated by miR-192 in NB cell lines. The relative luciferase unit (RLU) was measured in SHEP (A) or NUB (B) cells.A. The dual-luciferase assay resulted in a significant reduction of RLU of WT Dicer1 (3' UTR of Dicer1 wild type) following transfection with miR-192-vec (*p=0.049).B. Following transfection with miR-192 mimic, WT Dicer1 RLU was significantly decreased (*p=0.0003). Following mutagenesis, cells were transfected with Dicer1 plasmid in which mutations were introduced in all three BSs of Dicer1 (MUT ALL); active BS1 (mutated at BS2+BS3)(* p=0.004); active BS2 (mutated at BS1+BS3) (* p=0.04) and active BS3 (mutated at BS1+BS2). Values are expressed as the mean ± SE of combined results from three independent experiments.
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Related In: Results  -  Collection

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pone-0078713-g006: 3′UTR of Dicer 1 is directly targeted by miR-192.The dual luciferase assay detected that Dicer1 is modulated by miR-192 in NB cell lines. The relative luciferase unit (RLU) was measured in SHEP (A) or NUB (B) cells.A. The dual-luciferase assay resulted in a significant reduction of RLU of WT Dicer1 (3' UTR of Dicer1 wild type) following transfection with miR-192-vec (*p=0.049).B. Following transfection with miR-192 mimic, WT Dicer1 RLU was significantly decreased (*p=0.0003). Following mutagenesis, cells were transfected with Dicer1 plasmid in which mutations were introduced in all three BSs of Dicer1 (MUT ALL); active BS1 (mutated at BS2+BS3)(* p=0.004); active BS2 (mutated at BS1+BS3) (* p=0.04) and active BS3 (mutated at BS1+BS2). Values are expressed as the mean ± SE of combined results from three independent experiments.
Mentions: To validate the relative relevance of each one of the potential BSs of miR-192 in the 3′ untranslated region (3′UTR) of Dicer1, two different dual-luciferase reporter assays were carried out in NB cell lines. The dual-luciferase assay resulted in a significant reduction of 30% in the relative luciferase unit (RLU) of Dicer1 as was evident in SHEP cell line (non-MYCNA) transfected with miR-192-vec (p=0.049; Figure 6A). Moreover, a significant decrease of 59% in the level of Dicer1 RLU was evident in the NUB6 cell line (MYCNA) after transfection with the miR-192 mimic (p=0.0003; Figure 6B). Following transfection with miR-192 inhibitor the Dicer1 RLU returned to the control basal level (determined by transfection of scrambled miR sequences). These results suggest that miR-192 expression regulates Dicer1 protein expression.

Bottom Line: We were able to show through a dual luciferase assay and side-directed mutational analysis that miR-192 directly binds the 3' UTR of Dicer1 on positions 1232-1238 and 2282-2288.An increase in cell viability, proliferation and migration rates were evident in NB cells transfected with miR-192-mimic.Yet, there was a significant decrease in proliferation when NB cells were transfected with an miR-192-inhibitor We suggest that miR-192 might be a key player in NB by regulating Dicer1 expression.

View Article: PubMed Central - PubMed

Affiliation: Molecular Oncology, Felsenstein Medical Research Center, Petah Tikva, Israel ; Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.

ABSTRACT
Neuroblastoma (NB) arises from the embryonic neural crest and is the most common extracranial solid tumor in children under 5 years of age. Reduced expression of Dicer1 has recently been shown to be in correlation with poor prognosis in NB patients. This study aimed to investigate the mechanisms that could lead to the down-regulation of Dicer1 in neuroblastoma. We used computational prediction to identify potential miRs down-regulating Dicer1 in neuroblastoma. One of the miRs that were predicted to target Dicer1 was miR-192. We measured the levels of miR-192 in 43 primary tumors using real time PCR. Following the silencing of miR-192, the levels of dicer1 cell viability, cell proliferation and migration capability were analyzed. Multivariate analysis identified miR-192 as an independent prognostic marker for relapse in neuroblastoma patients (p=0.04). We were able to show through a dual luciferase assay and side-directed mutational analysis that miR-192 directly binds the 3' UTR of Dicer1 on positions 1232-1238 and 2282-2288. An increase in cell viability, proliferation and migration rates were evident in NB cells transfected with miR-192-mimic. Yet, there was a significant decrease in proliferation when NB cells were transfected with an miR-192-inhibitor We suggest that miR-192 might be a key player in NB by regulating Dicer1 expression.

Show MeSH
Related in: MedlinePlus