MIR-181C REGULATES BRK1, PART OF THE WAVE COMPLEX, AND IS INVOLVED IN T CELL FUNCTION
(Abstract release date: 05/19/16)
EHA Library. Lim S. 06/09/16; 132945; E1396
Ms. Shok Ping Lim
Contributions
Contributions
Abstract
Abstract: E1396
Type: Eposter Presentation
Background
miRNAs are short endogenous non-coding RNAs of 18-25 nucleotides in length which play an important role as negative regulators of gene expression either through mRNA degradation or translational repression. miRNAs have been shown to be involved in many cell functions such as angiogenesis, cell adhesion, cell differentiation, cell proliferation and cell survival. Thus, their aberrant expression implicated in various types of cancer including haematological malignancies. In this project, we studied miR-181 family which has been reported to play a crucial role in haematopoiesis, including megakaryocytic, erythroid and myeloid differentiation as well as B and T cell development and differentiation.Most of the available methodologies for miRNA target identification are based on computational algorithms. Therefore, there is a relatively high incidence of false positive target identification. To enable the identification of biologically relevant miRNA targets, a novel functional assay was developed in our lab, which is based on positive/negative selection (Gaken et al, 2012). By using this assay, we identified two functional targets of miR-181c which are BRK1 and DHTKD1.Due to the well-known role of WAVE protein (BRK1 is a component of the WAVE complex) in actin polymerisation in T cells, and the published studies on the function of BRK1 in cell migration in neuroblastoma, squamous cell lung carcinoma, renal carcinoma and osteosarcoma, we focused on studying the biological roles of BRK1 in T cells. As a model system, we used Jurkat T cells and investigated the role of BRK1 in actin polymerisation.
Aims
The aims of this project are to identify the potential targets of miR-181 family by using a dual selection functional assay and to study the roles of BRK1 (identified target of miR-181c) in actin polymerisation and T cell functions, in association with WAVE2 complex.
Methods
Using a functional assay, we identified BRK1 and DHTKD1 as novel targets of miR-181c/d and validated these targets by quantitative RT-PCR and western blot analysis of cells transfected with miR-181c/d as well as miR-181c and d inhibitors. We focused on ascertaining the roles of BRK1 in T cell functions. We demonstrated a targeted reduction of BRK1 in Jurkat T cells through shRNA (lentiviral strategy) or siRNA knockdown. The effects of BRK1 knockdown on actin polymerisation and Jurkat T cells functions including changes in total cellular F-actin content, T cell proliferation, T cell activation, lamellipodia formation and T cell spreading as well as cell migration were assessed.
Results
By using the novel functional assay, two targets of miR-181c/d have been identified which are BRK1 and DHTKD1. Both of the genes were validated as targets for miR-181c at protein level. To study the roles of BRK1 in Jurkat T cell functions, we first showed that knockdown of BRK1 in Jurkat T cells reduced the expression of other proteins (WAVE2, Abi1, Sra1 and Hem1) that are part of the WAVE2 complex and this indicated that any effects on T cell functions due to BRK1 knockdown is indeed a result of dissociation of WAVE2 complex. Stable knockdown of BRK1 in Jurkat T cells showed changes in total cellular F-actin content prior and after T cell activation by F-actin staining using Rhodamine phalloidin. Next, we showed that stable BRK1 knockdown decreased cell proliferation by trypan blue staining. Also, BRK1 knockdown showed reduced CD69 expression, indicating defects in T cell activation. Besides that, we also demonstrated that knockdown of BRK1 caused defects in lamellipodia formation and cell spreading upon Jurkat T cell activation. Knockdown of BRK1 also resulted in defects in cell migration in Jurkat T cells.
Conclusion
This study showed that miR-181c regulates BRK1 by translational repression and BRK1 plays a crucial role in Jurkat T cell functions including cell proliferation, T cell activation, cell spreading and cell migration mediated via actin polymerisation.
Session topic: E-poster
Keyword(s): T cell
Type: Eposter Presentation
Background
miRNAs are short endogenous non-coding RNAs of 18-25 nucleotides in length which play an important role as negative regulators of gene expression either through mRNA degradation or translational repression. miRNAs have been shown to be involved in many cell functions such as angiogenesis, cell adhesion, cell differentiation, cell proliferation and cell survival. Thus, their aberrant expression implicated in various types of cancer including haematological malignancies. In this project, we studied miR-181 family which has been reported to play a crucial role in haematopoiesis, including megakaryocytic, erythroid and myeloid differentiation as well as B and T cell development and differentiation.Most of the available methodologies for miRNA target identification are based on computational algorithms. Therefore, there is a relatively high incidence of false positive target identification. To enable the identification of biologically relevant miRNA targets, a novel functional assay was developed in our lab, which is based on positive/negative selection (Gaken et al, 2012). By using this assay, we identified two functional targets of miR-181c which are BRK1 and DHTKD1.Due to the well-known role of WAVE protein (BRK1 is a component of the WAVE complex) in actin polymerisation in T cells, and the published studies on the function of BRK1 in cell migration in neuroblastoma, squamous cell lung carcinoma, renal carcinoma and osteosarcoma, we focused on studying the biological roles of BRK1 in T cells. As a model system, we used Jurkat T cells and investigated the role of BRK1 in actin polymerisation.
Aims
The aims of this project are to identify the potential targets of miR-181 family by using a dual selection functional assay and to study the roles of BRK1 (identified target of miR-181c) in actin polymerisation and T cell functions, in association with WAVE2 complex.
Methods
Using a functional assay, we identified BRK1 and DHTKD1 as novel targets of miR-181c/d and validated these targets by quantitative RT-PCR and western blot analysis of cells transfected with miR-181c/d as well as miR-181c and d inhibitors. We focused on ascertaining the roles of BRK1 in T cell functions. We demonstrated a targeted reduction of BRK1 in Jurkat T cells through shRNA (lentiviral strategy) or siRNA knockdown. The effects of BRK1 knockdown on actin polymerisation and Jurkat T cells functions including changes in total cellular F-actin content, T cell proliferation, T cell activation, lamellipodia formation and T cell spreading as well as cell migration were assessed.
Results
By using the novel functional assay, two targets of miR-181c/d have been identified which are BRK1 and DHTKD1. Both of the genes were validated as targets for miR-181c at protein level. To study the roles of BRK1 in Jurkat T cell functions, we first showed that knockdown of BRK1 in Jurkat T cells reduced the expression of other proteins (WAVE2, Abi1, Sra1 and Hem1) that are part of the WAVE2 complex and this indicated that any effects on T cell functions due to BRK1 knockdown is indeed a result of dissociation of WAVE2 complex. Stable knockdown of BRK1 in Jurkat T cells showed changes in total cellular F-actin content prior and after T cell activation by F-actin staining using Rhodamine phalloidin. Next, we showed that stable BRK1 knockdown decreased cell proliferation by trypan blue staining. Also, BRK1 knockdown showed reduced CD69 expression, indicating defects in T cell activation. Besides that, we also demonstrated that knockdown of BRK1 caused defects in lamellipodia formation and cell spreading upon Jurkat T cell activation. Knockdown of BRK1 also resulted in defects in cell migration in Jurkat T cells.
Conclusion
This study showed that miR-181c regulates BRK1 by translational repression and BRK1 plays a crucial role in Jurkat T cell functions including cell proliferation, T cell activation, cell spreading and cell migration mediated via actin polymerisation.
Session topic: E-poster
Keyword(s): T cell
Abstract: E1396
Type: Eposter Presentation
Background
miRNAs are short endogenous non-coding RNAs of 18-25 nucleotides in length which play an important role as negative regulators of gene expression either through mRNA degradation or translational repression. miRNAs have been shown to be involved in many cell functions such as angiogenesis, cell adhesion, cell differentiation, cell proliferation and cell survival. Thus, their aberrant expression implicated in various types of cancer including haematological malignancies. In this project, we studied miR-181 family which has been reported to play a crucial role in haematopoiesis, including megakaryocytic, erythroid and myeloid differentiation as well as B and T cell development and differentiation.Most of the available methodologies for miRNA target identification are based on computational algorithms. Therefore, there is a relatively high incidence of false positive target identification. To enable the identification of biologically relevant miRNA targets, a novel functional assay was developed in our lab, which is based on positive/negative selection (Gaken et al, 2012). By using this assay, we identified two functional targets of miR-181c which are BRK1 and DHTKD1.Due to the well-known role of WAVE protein (BRK1 is a component of the WAVE complex) in actin polymerisation in T cells, and the published studies on the function of BRK1 in cell migration in neuroblastoma, squamous cell lung carcinoma, renal carcinoma and osteosarcoma, we focused on studying the biological roles of BRK1 in T cells. As a model system, we used Jurkat T cells and investigated the role of BRK1 in actin polymerisation.
Aims
The aims of this project are to identify the potential targets of miR-181 family by using a dual selection functional assay and to study the roles of BRK1 (identified target of miR-181c) in actin polymerisation and T cell functions, in association with WAVE2 complex.
Methods
Using a functional assay, we identified BRK1 and DHTKD1 as novel targets of miR-181c/d and validated these targets by quantitative RT-PCR and western blot analysis of cells transfected with miR-181c/d as well as miR-181c and d inhibitors. We focused on ascertaining the roles of BRK1 in T cell functions. We demonstrated a targeted reduction of BRK1 in Jurkat T cells through shRNA (lentiviral strategy) or siRNA knockdown. The effects of BRK1 knockdown on actin polymerisation and Jurkat T cells functions including changes in total cellular F-actin content, T cell proliferation, T cell activation, lamellipodia formation and T cell spreading as well as cell migration were assessed.
Results
By using the novel functional assay, two targets of miR-181c/d have been identified which are BRK1 and DHTKD1. Both of the genes were validated as targets for miR-181c at protein level. To study the roles of BRK1 in Jurkat T cell functions, we first showed that knockdown of BRK1 in Jurkat T cells reduced the expression of other proteins (WAVE2, Abi1, Sra1 and Hem1) that are part of the WAVE2 complex and this indicated that any effects on T cell functions due to BRK1 knockdown is indeed a result of dissociation of WAVE2 complex. Stable knockdown of BRK1 in Jurkat T cells showed changes in total cellular F-actin content prior and after T cell activation by F-actin staining using Rhodamine phalloidin. Next, we showed that stable BRK1 knockdown decreased cell proliferation by trypan blue staining. Also, BRK1 knockdown showed reduced CD69 expression, indicating defects in T cell activation. Besides that, we also demonstrated that knockdown of BRK1 caused defects in lamellipodia formation and cell spreading upon Jurkat T cell activation. Knockdown of BRK1 also resulted in defects in cell migration in Jurkat T cells.
Conclusion
This study showed that miR-181c regulates BRK1 by translational repression and BRK1 plays a crucial role in Jurkat T cell functions including cell proliferation, T cell activation, cell spreading and cell migration mediated via actin polymerisation.
Session topic: E-poster
Keyword(s): T cell
Type: Eposter Presentation
Background
miRNAs are short endogenous non-coding RNAs of 18-25 nucleotides in length which play an important role as negative regulators of gene expression either through mRNA degradation or translational repression. miRNAs have been shown to be involved in many cell functions such as angiogenesis, cell adhesion, cell differentiation, cell proliferation and cell survival. Thus, their aberrant expression implicated in various types of cancer including haematological malignancies. In this project, we studied miR-181 family which has been reported to play a crucial role in haematopoiesis, including megakaryocytic, erythroid and myeloid differentiation as well as B and T cell development and differentiation.Most of the available methodologies for miRNA target identification are based on computational algorithms. Therefore, there is a relatively high incidence of false positive target identification. To enable the identification of biologically relevant miRNA targets, a novel functional assay was developed in our lab, which is based on positive/negative selection (Gaken et al, 2012). By using this assay, we identified two functional targets of miR-181c which are BRK1 and DHTKD1.Due to the well-known role of WAVE protein (BRK1 is a component of the WAVE complex) in actin polymerisation in T cells, and the published studies on the function of BRK1 in cell migration in neuroblastoma, squamous cell lung carcinoma, renal carcinoma and osteosarcoma, we focused on studying the biological roles of BRK1 in T cells. As a model system, we used Jurkat T cells and investigated the role of BRK1 in actin polymerisation.
Aims
The aims of this project are to identify the potential targets of miR-181 family by using a dual selection functional assay and to study the roles of BRK1 (identified target of miR-181c) in actin polymerisation and T cell functions, in association with WAVE2 complex.
Methods
Using a functional assay, we identified BRK1 and DHTKD1 as novel targets of miR-181c/d and validated these targets by quantitative RT-PCR and western blot analysis of cells transfected with miR-181c/d as well as miR-181c and d inhibitors. We focused on ascertaining the roles of BRK1 in T cell functions. We demonstrated a targeted reduction of BRK1 in Jurkat T cells through shRNA (lentiviral strategy) or siRNA knockdown. The effects of BRK1 knockdown on actin polymerisation and Jurkat T cells functions including changes in total cellular F-actin content, T cell proliferation, T cell activation, lamellipodia formation and T cell spreading as well as cell migration were assessed.
Results
By using the novel functional assay, two targets of miR-181c/d have been identified which are BRK1 and DHTKD1. Both of the genes were validated as targets for miR-181c at protein level. To study the roles of BRK1 in Jurkat T cell functions, we first showed that knockdown of BRK1 in Jurkat T cells reduced the expression of other proteins (WAVE2, Abi1, Sra1 and Hem1) that are part of the WAVE2 complex and this indicated that any effects on T cell functions due to BRK1 knockdown is indeed a result of dissociation of WAVE2 complex. Stable knockdown of BRK1 in Jurkat T cells showed changes in total cellular F-actin content prior and after T cell activation by F-actin staining using Rhodamine phalloidin. Next, we showed that stable BRK1 knockdown decreased cell proliferation by trypan blue staining. Also, BRK1 knockdown showed reduced CD69 expression, indicating defects in T cell activation. Besides that, we also demonstrated that knockdown of BRK1 caused defects in lamellipodia formation and cell spreading upon Jurkat T cell activation. Knockdown of BRK1 also resulted in defects in cell migration in Jurkat T cells.
Conclusion
This study showed that miR-181c regulates BRK1 by translational repression and BRK1 plays a crucial role in Jurkat T cell functions including cell proliferation, T cell activation, cell spreading and cell migration mediated via actin polymerisation.
Session topic: E-poster
Keyword(s): T cell
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