Limits...
Metastasis suppressor microRNA-335 targets the formin family of actin nucleators.

Lynch J, Meehan MH, Crean J, Copeland J, Stallings RL, Bray IM - PLoS ONE (2013)

Bottom Line: MiRNAs can have pleiotropic effects by targeting multiple genes belonging to diverse signalling networks.Finally, we demonstrate that the formin genes, in particular FMNL3, are responsible for the protrusion of actin-rich filopodia structures that contribute to the enhanced migratory and invasive potential associated with reduced expression of miR-335.We conclude that miR-335 is a master regulator of tumour cell migration and invasion by directly targeting a plethora of genes that effectively control cell migratory processes.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Genetics, Royal College of Surgeons in Ireland, Dublin, Ireland ; National Children's Research Centre, Our Lady's Children's Hospital, Dublin, Ireland.

ABSTRACT
MiRNAs can have pleiotropic effects by targeting multiple genes belonging to diverse signalling networks. Alternatively, miRNAs can enhance the potency of their cellular effects by targeting multiple genes within the same genetic pathway. Previously, we and others have demonstrated that miR-335 is a potent suppressor of tumour cell migration, invasion and metastasis, in part by targeting several genes involved in these cellular processes, including ROCK1, MAPK1, LRG1, SP1 and SOX4. Here, we demonstrate that direct targeting of multiple members of the formin family of actin nucleators contributes to the inhibitory effects of miR-335 in neuroblastoma cells. We demonstrate that miR-335 regulates the expression of at least five formin family members and validate three family members, FMNL3, FMN2 and DAAM2, as direct targets of miR-335. The contribution of the formin family genes to cancer progression and metastasis has recently begun to emerge and here we demonstrate for the first time the ability of FMN2 and DAAM2 to regulate tumour cell migration and invasion, using siRNA-mediated inhibition of each of these formin genes. Finally, we demonstrate that the formin genes, in particular FMNL3, are responsible for the protrusion of actin-rich filopodia structures that contribute to the enhanced migratory and invasive potential associated with reduced expression of miR-335. Thus, direct targeting of the formin family contributes to the metastasis suppressing abilities of miR-335 by providing a direct regulatory link to the actin assembly machinery of the cell. We conclude that miR-335 is a master regulator of tumour cell migration and invasion by directly targeting a plethora of genes that effectively control cell migratory processes.

Show MeSH

Related in: MedlinePlus

MiR-335, FMNL3, FMN2 and DAAM2 regulate actin cytoskeleton dynamics.(a) Transfection of cells with miR-335 mimics resulted in a reduction in the levels of phosphorylated cofilin protein and did not affect the levels of total cofilin protein (b) siRNA-mediated inhibition of FMNL3, FMN2 and DAAM2 produced a reduction in phosphorylated cofilin levels without altering total cofilin protein levels. Densitometric quantification of reduced phospho-cofilin protein in response to enhanced miR-335 expression (c) and reduced expression of FMNL3, FMN2 and DAAM2 (d) performed on duplicate blots and normalised against the appropriate alpha-tubulin loading control.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3818330&req=5

pone-0078428-g005: MiR-335, FMNL3, FMN2 and DAAM2 regulate actin cytoskeleton dynamics.(a) Transfection of cells with miR-335 mimics resulted in a reduction in the levels of phosphorylated cofilin protein and did not affect the levels of total cofilin protein (b) siRNA-mediated inhibition of FMNL3, FMN2 and DAAM2 produced a reduction in phosphorylated cofilin levels without altering total cofilin protein levels. Densitometric quantification of reduced phospho-cofilin protein in response to enhanced miR-335 expression (c) and reduced expression of FMNL3, FMN2 and DAAM2 (d) performed on duplicate blots and normalised against the appropriate alpha-tubulin loading control.

Mentions: Cofilin, a member of the actin depolymerisation factor (ADF) family, plays a key role in controlling actin filament dynamics and reorganisation by depolymerising and severing actin filaments [23], [24]. Given its role as a critical regulator of actin dynamics, cofilin can be used as a marker for assessing alterations to actin cytoskeleton dynamics. The actin depolymerising activities of cofilin are subject to negative regulation by phosphorylation of Ser-3 by LIM kinase [25], [26]. The phosphorylation status of cofilin was assessed to determine if actin cytoskeleton dynamics were disrupted as a consequence of altered miR-335 expression and subsequent formin target gene expression. CHP-212 cells were transfected with miR-335 mimics and the phosphorylation levels of cofilin were analysed by Western blot 24 hours post-transfection using a phospho antibody specific to residue Ser-3 of cofilin. As illustrated in Figure 5a, miR-335 up-regulation stimulated a resultant reduction in the levels of phosphorylated cofilin protein compared to negative control cells, while not affecting total levels of cofilin protein. Correspondingly, siRNA-mediated knockdown of each of the three formin genes FMNL3, FMN2 and DAAM2 also resulted in reduced phosphorylation levels of cofilin (without affecting total protein levels), compared to negative control cells 24 hours post-transfection (Figure 5b). Significant reductions in phosphophorylated cofilin levels were quantified by densitometric analysis as displayed for miR-335 transfected cells (Figure 5c) and cells transfected with siRNAs to FMNL3, FMN2 and DAAM2 (Figure 5d), full western blot images displayed in Supplemental Figure S5. This would suggest that disruption to actin dynamics contributes to the reduced migratory capacity associated with the up-regulated expression of miR-335 and down-regulated expression of FMNL3, FMN2 and DAAM2.


Metastasis suppressor microRNA-335 targets the formin family of actin nucleators.

Lynch J, Meehan MH, Crean J, Copeland J, Stallings RL, Bray IM - PLoS ONE (2013)

MiR-335, FMNL3, FMN2 and DAAM2 regulate actin cytoskeleton dynamics.(a) Transfection of cells with miR-335 mimics resulted in a reduction in the levels of phosphorylated cofilin protein and did not affect the levels of total cofilin protein (b) siRNA-mediated inhibition of FMNL3, FMN2 and DAAM2 produced a reduction in phosphorylated cofilin levels without altering total cofilin protein levels. Densitometric quantification of reduced phospho-cofilin protein in response to enhanced miR-335 expression (c) and reduced expression of FMNL3, FMN2 and DAAM2 (d) performed on duplicate blots and normalised against the appropriate alpha-tubulin loading control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3818330&req=5

pone-0078428-g005: MiR-335, FMNL3, FMN2 and DAAM2 regulate actin cytoskeleton dynamics.(a) Transfection of cells with miR-335 mimics resulted in a reduction in the levels of phosphorylated cofilin protein and did not affect the levels of total cofilin protein (b) siRNA-mediated inhibition of FMNL3, FMN2 and DAAM2 produced a reduction in phosphorylated cofilin levels without altering total cofilin protein levels. Densitometric quantification of reduced phospho-cofilin protein in response to enhanced miR-335 expression (c) and reduced expression of FMNL3, FMN2 and DAAM2 (d) performed on duplicate blots and normalised against the appropriate alpha-tubulin loading control.
Mentions: Cofilin, a member of the actin depolymerisation factor (ADF) family, plays a key role in controlling actin filament dynamics and reorganisation by depolymerising and severing actin filaments [23], [24]. Given its role as a critical regulator of actin dynamics, cofilin can be used as a marker for assessing alterations to actin cytoskeleton dynamics. The actin depolymerising activities of cofilin are subject to negative regulation by phosphorylation of Ser-3 by LIM kinase [25], [26]. The phosphorylation status of cofilin was assessed to determine if actin cytoskeleton dynamics were disrupted as a consequence of altered miR-335 expression and subsequent formin target gene expression. CHP-212 cells were transfected with miR-335 mimics and the phosphorylation levels of cofilin were analysed by Western blot 24 hours post-transfection using a phospho antibody specific to residue Ser-3 of cofilin. As illustrated in Figure 5a, miR-335 up-regulation stimulated a resultant reduction in the levels of phosphorylated cofilin protein compared to negative control cells, while not affecting total levels of cofilin protein. Correspondingly, siRNA-mediated knockdown of each of the three formin genes FMNL3, FMN2 and DAAM2 also resulted in reduced phosphorylation levels of cofilin (without affecting total protein levels), compared to negative control cells 24 hours post-transfection (Figure 5b). Significant reductions in phosphophorylated cofilin levels were quantified by densitometric analysis as displayed for miR-335 transfected cells (Figure 5c) and cells transfected with siRNAs to FMNL3, FMN2 and DAAM2 (Figure 5d), full western blot images displayed in Supplemental Figure S5. This would suggest that disruption to actin dynamics contributes to the reduced migratory capacity associated with the up-regulated expression of miR-335 and down-regulated expression of FMNL3, FMN2 and DAAM2.

Bottom Line: MiRNAs can have pleiotropic effects by targeting multiple genes belonging to diverse signalling networks.Finally, we demonstrate that the formin genes, in particular FMNL3, are responsible for the protrusion of actin-rich filopodia structures that contribute to the enhanced migratory and invasive potential associated with reduced expression of miR-335.We conclude that miR-335 is a master regulator of tumour cell migration and invasion by directly targeting a plethora of genes that effectively control cell migratory processes.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Genetics, Royal College of Surgeons in Ireland, Dublin, Ireland ; National Children's Research Centre, Our Lady's Children's Hospital, Dublin, Ireland.

ABSTRACT
MiRNAs can have pleiotropic effects by targeting multiple genes belonging to diverse signalling networks. Alternatively, miRNAs can enhance the potency of their cellular effects by targeting multiple genes within the same genetic pathway. Previously, we and others have demonstrated that miR-335 is a potent suppressor of tumour cell migration, invasion and metastasis, in part by targeting several genes involved in these cellular processes, including ROCK1, MAPK1, LRG1, SP1 and SOX4. Here, we demonstrate that direct targeting of multiple members of the formin family of actin nucleators contributes to the inhibitory effects of miR-335 in neuroblastoma cells. We demonstrate that miR-335 regulates the expression of at least five formin family members and validate three family members, FMNL3, FMN2 and DAAM2, as direct targets of miR-335. The contribution of the formin family genes to cancer progression and metastasis has recently begun to emerge and here we demonstrate for the first time the ability of FMN2 and DAAM2 to regulate tumour cell migration and invasion, using siRNA-mediated inhibition of each of these formin genes. Finally, we demonstrate that the formin genes, in particular FMNL3, are responsible for the protrusion of actin-rich filopodia structures that contribute to the enhanced migratory and invasive potential associated with reduced expression of miR-335. Thus, direct targeting of the formin family contributes to the metastasis suppressing abilities of miR-335 by providing a direct regulatory link to the actin assembly machinery of the cell. We conclude that miR-335 is a master regulator of tumour cell migration and invasion by directly targeting a plethora of genes that effectively control cell migratory processes.

Show MeSH
Related in: MedlinePlus