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Regulation of a TrkB Alternative Transcript by microRNAs.

Wong J - Dement Geriatr Cogn Dis Extra (2014)

Bottom Line: A combination of bioinformatics and molecular gene expression analysis techniques was used to assess the effect of miR-409-3p and miR-216b on TrkB-Shc expression. miR-409-3p and miR-216b were found to regulate the TrkB-Shc 3'UTR through the identified putative binding sites.When the effect of the miRNAs on TrkB was assessed using SHSY5Y neuronal cells, differential effects were observed between mRNA and protein expression.This study highlights the importance of miRNA-mediated regulation in TrkB signaling.

View Article: PubMed Central - PubMed

Affiliation: Illawarra Health and Medical Research Institute and School of Biological Sciences, University of Wollongong, Wollongong, N.S.W., Australia.

ABSTRACT

Background/aims: Tropomyosin-related kinase B receptor (TrkB)-mediated signaling is vital for neuronal differentiation, survival, plasticity, and cognition. In this study, the focus was placed on TrkB-Shc, a neuron-specific transcript, to determine if microRNAs (miRNAs) play a role in TrkB-Shc regulation.

Methods: A combination of bioinformatics and molecular gene expression analysis techniques was used to assess the effect of miR-409-3p and miR-216b on TrkB-Shc expression.

Results: miR-409-3p and miR-216b were found to regulate the TrkB-Shc 3'UTR through the identified putative binding sites. When the effect of the miRNAs on TrkB was assessed using SHSY5Y neuronal cells, differential effects were observed between mRNA and protein expression.

Conclusion: This study highlights the importance of miRNA-mediated regulation in TrkB signaling.

No MeSH data available.


Related in: MedlinePlus

Regulation of the TrkB-Shc 3′UTR by miR-409-3p and miR-216b. HEK293 cells were cotransfected with pGL3-SV40 (empty vector; white column) or a pGL3-SV40 construct containing the TrkB-Shc 3′UTR (black column) in combination with a miR-409-3p or control miRNA and b miR-216b or control miRNA. Data are normalized to the respective empty vector controls and expressed as relative luciferase activity. Values are presented as mean ± SEM and are representative of n = 3 independent experiments from three replicate cultures. * p = 0.002; ** p = 0.00002.
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Figure 2: Regulation of the TrkB-Shc 3′UTR by miR-409-3p and miR-216b. HEK293 cells were cotransfected with pGL3-SV40 (empty vector; white column) or a pGL3-SV40 construct containing the TrkB-Shc 3′UTR (black column) in combination with a miR-409-3p or control miRNA and b miR-216b or control miRNA. Data are normalized to the respective empty vector controls and expressed as relative luciferase activity. Values are presented as mean ± SEM and are representative of n = 3 independent experiments from three replicate cultures. * p = 0.002; ** p = 0.00002.

Mentions: It was next determined whether miR-409-3p and miR-216b could regulate TrkB-Shc transcript expression through its 3′UTR. The TrkB-Shc 3′UTR was cloned into the 3′UTR of the pGL3-SV40 luciferase reporter vector. Then, HEK293 cells were cotransfected with either this luciferase reporter construct or an empty vector with either miR-409-3p, miR-216b, or control miRNA. Highly significant decreases in luciferase reporter activity were observed following miR-409-3p and miR-216b transfection (miR-409-3p: t = 7.10, d.f. = 4, p = 0.002; miR-216b: t = 23.3, d.f. = 4, p = 0.00002) (fig. 2a, b). No significant changes were observed in the empty vector control transfections (miR-409-3p: t = −2.07, d.f. = 4, p = 0.0.11; miR-216b: t = −1.87, d.f. = 4, p = 0.14) (fig. 2a, b). Consistent with observations using only the putative mi409-3p- and miR-216b-binding sites, it was found that both miR-409-3p and miR-216b could decrease luciferase reporter activity in cells transfected with constructs containing the endogenous TrkB-Shc 3′UTR.


Regulation of a TrkB Alternative Transcript by microRNAs.

Wong J - Dement Geriatr Cogn Dis Extra (2014)

Regulation of the TrkB-Shc 3′UTR by miR-409-3p and miR-216b. HEK293 cells were cotransfected with pGL3-SV40 (empty vector; white column) or a pGL3-SV40 construct containing the TrkB-Shc 3′UTR (black column) in combination with a miR-409-3p or control miRNA and b miR-216b or control miRNA. Data are normalized to the respective empty vector controls and expressed as relative luciferase activity. Values are presented as mean ± SEM and are representative of n = 3 independent experiments from three replicate cultures. * p = 0.002; ** p = 0.00002.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4202609&req=5

Figure 2: Regulation of the TrkB-Shc 3′UTR by miR-409-3p and miR-216b. HEK293 cells were cotransfected with pGL3-SV40 (empty vector; white column) or a pGL3-SV40 construct containing the TrkB-Shc 3′UTR (black column) in combination with a miR-409-3p or control miRNA and b miR-216b or control miRNA. Data are normalized to the respective empty vector controls and expressed as relative luciferase activity. Values are presented as mean ± SEM and are representative of n = 3 independent experiments from three replicate cultures. * p = 0.002; ** p = 0.00002.
Mentions: It was next determined whether miR-409-3p and miR-216b could regulate TrkB-Shc transcript expression through its 3′UTR. The TrkB-Shc 3′UTR was cloned into the 3′UTR of the pGL3-SV40 luciferase reporter vector. Then, HEK293 cells were cotransfected with either this luciferase reporter construct or an empty vector with either miR-409-3p, miR-216b, or control miRNA. Highly significant decreases in luciferase reporter activity were observed following miR-409-3p and miR-216b transfection (miR-409-3p: t = 7.10, d.f. = 4, p = 0.002; miR-216b: t = 23.3, d.f. = 4, p = 0.00002) (fig. 2a, b). No significant changes were observed in the empty vector control transfections (miR-409-3p: t = −2.07, d.f. = 4, p = 0.0.11; miR-216b: t = −1.87, d.f. = 4, p = 0.14) (fig. 2a, b). Consistent with observations using only the putative mi409-3p- and miR-216b-binding sites, it was found that both miR-409-3p and miR-216b could decrease luciferase reporter activity in cells transfected with constructs containing the endogenous TrkB-Shc 3′UTR.

Bottom Line: A combination of bioinformatics and molecular gene expression analysis techniques was used to assess the effect of miR-409-3p and miR-216b on TrkB-Shc expression. miR-409-3p and miR-216b were found to regulate the TrkB-Shc 3'UTR through the identified putative binding sites.When the effect of the miRNAs on TrkB was assessed using SHSY5Y neuronal cells, differential effects were observed between mRNA and protein expression.This study highlights the importance of miRNA-mediated regulation in TrkB signaling.

View Article: PubMed Central - PubMed

Affiliation: Illawarra Health and Medical Research Institute and School of Biological Sciences, University of Wollongong, Wollongong, N.S.W., Australia.

ABSTRACT

Background/aims: Tropomyosin-related kinase B receptor (TrkB)-mediated signaling is vital for neuronal differentiation, survival, plasticity, and cognition. In this study, the focus was placed on TrkB-Shc, a neuron-specific transcript, to determine if microRNAs (miRNAs) play a role in TrkB-Shc regulation.

Methods: A combination of bioinformatics and molecular gene expression analysis techniques was used to assess the effect of miR-409-3p and miR-216b on TrkB-Shc expression.

Results: miR-409-3p and miR-216b were found to regulate the TrkB-Shc 3'UTR through the identified putative binding sites. When the effect of the miRNAs on TrkB was assessed using SHSY5Y neuronal cells, differential effects were observed between mRNA and protein expression.

Conclusion: This study highlights the importance of miRNA-mediated regulation in TrkB signaling.

No MeSH data available.


Related in: MedlinePlus