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Detrusor induction of miR-132/212 following bladder outlet obstruction: association with MeCP2 repression and cell viability.

Sadegh MK, Ekman M, Krawczyk K, Svensson D, Göransson O, Dahan D, Nilsson BO, Albinsson S, Uvelius B, Swärd K - PLoS ONE (2015)

Bottom Line: The microRNAs (miRNAs) miR-132 and miR-212 have been found to regulate synaptic plasticity and cholinergic signaling and recent work has demonstrated roles outside of the CNS, including in smooth muscle.Moreover, we found that serum stimulation and protein kinase C activation induced miR-132/212 in human detrusor cells.Importantly, transfection of antimirs and mimics in cultured detrusor cells increased and decreased, respectively, the number of cells and led to changes in MeCP2 expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Medical Science, Lund University, Lund, Sweden.

ABSTRACT
The microRNAs (miRNAs) miR-132 and miR-212 have been found to regulate synaptic plasticity and cholinergic signaling and recent work has demonstrated roles outside of the CNS, including in smooth muscle. Here, we examined if miR-132 and miR-212 are induced in the urinary bladder following outlet obstruction and whether this correlates with effects on gene expression and cell growth. Three to seven-fold induction of miR-132/212 was found at 10 days of obstruction and this was selective for the detrusor layer. We cross-referenced putative binding sites in the miR-132/212 promoter with transcription factors that were predicted to be active in the obstruction model. This suggested involvement of Creb and Ahr in miR-132/212 induction. Creb phosphorylation (S-133) was not increased, but the number of Ahr positive nuclei increased. Moreover, we found that serum stimulation and protein kinase C activation induced miR-132/212 in human detrusor cells. To identify miR-132/212 targets, we correlated the mRNA levels of validated targets with the miRNA levels. Significant correlations between miR-132/212 and MeCP2, Ep300, Pnkd and Jarid1a were observed, and the protein levels of MeCP2, Pnkd and Ache were reduced after obstruction. Reduction of Ache however closely matched a 90% reduction of synapse density arguing that its repression was unrelated to miR-132/212 induction. Importantly, transfection of antimirs and mimics in cultured detrusor cells increased and decreased, respectively, the number of cells and led to changes in MeCP2 expression. In all, these findings show that obstruction of the urethra increases miR-132 and miR-212 in the detrusor and suggests that this influences gene expression and limits cell growth.

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Transfection of miR-132/212 mimics and inhibitors affect MeCP2 expression and cell number in vitro.An initial dosing experiment performed in duplicate using human detrusor smooth muscle cells from one individual shows a reciprocal effect of miR-132 mimic and inhibitor on MeCP2 expression (A). The effect of 100 nM miR-132 mimic was confirmed using cells from three individuals (B). Panel C shows effect of miR-212 mimic and inhibitor (100 nM of each) on cell viability using the MTT assay. Panel D shows the effect of miR-212 mimic and inhibitor on cell number and panel E shows phase contrast light microscopic images of cells transfected with negative control (NC), miR-212 mimic and miR-212 inhibitor. The scale bar in E indicates 20 μm and n = 5 and 8 in C and D, respectively.
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pone.0116784.g007: Transfection of miR-132/212 mimics and inhibitors affect MeCP2 expression and cell number in vitro.An initial dosing experiment performed in duplicate using human detrusor smooth muscle cells from one individual shows a reciprocal effect of miR-132 mimic and inhibitor on MeCP2 expression (A). The effect of 100 nM miR-132 mimic was confirmed using cells from three individuals (B). Panel C shows effect of miR-212 mimic and inhibitor (100 nM of each) on cell viability using the MTT assay. Panel D shows the effect of miR-212 mimic and inhibitor on cell number and panel E shows phase contrast light microscopic images of cells transfected with negative control (NC), miR-212 mimic and miR-212 inhibitor. The scale bar in E indicates 20 μm and n = 5 and 8 in C and D, respectively.

Mentions: The lack of support for a role of miR-132/212 in regulation of Ache in the bladder led us to consider alternative roles. Using cultured human detrusor myocytes we transfected miR-132/212 mimics and antimirs. In a dosing experiment we found reciprocal changes in expression of the transcriptional regulator MeCP2 following transfection of miR-132 mimic and antimir (Fig. 7A). A similar trend was seen for miR-212 (not shown). The effect of 100 nM miR-132 mimic on MeCP2 expression was confirmed using cells from three different patients (Fig. 7B). We next examined effects on cell viability and cell number. MiR-212 mimic was found to reduce both viability and cell number (Fig. 7C and D), whereas the inhibitor increased cell number (Fig. 7D). These effects were apparent in bright field images of cells after transfection of miR-212 mimic and inhibitor compared to negative control (NC, Fig. 7E). Signs of apoptosis, such as cell membrane blebbing and cell shrinkage, were not apparent (Fig. 7E). MiR-132 inhibitor increased cell viability (+6.7±1.36%, p<0.01 vs. negative control), while miR-132 mimic was without effect (−0.7±1.6%, p = 0.75).


Detrusor induction of miR-132/212 following bladder outlet obstruction: association with MeCP2 repression and cell viability.

Sadegh MK, Ekman M, Krawczyk K, Svensson D, Göransson O, Dahan D, Nilsson BO, Albinsson S, Uvelius B, Swärd K - PLoS ONE (2015)

Transfection of miR-132/212 mimics and inhibitors affect MeCP2 expression and cell number in vitro.An initial dosing experiment performed in duplicate using human detrusor smooth muscle cells from one individual shows a reciprocal effect of miR-132 mimic and inhibitor on MeCP2 expression (A). The effect of 100 nM miR-132 mimic was confirmed using cells from three individuals (B). Panel C shows effect of miR-212 mimic and inhibitor (100 nM of each) on cell viability using the MTT assay. Panel D shows the effect of miR-212 mimic and inhibitor on cell number and panel E shows phase contrast light microscopic images of cells transfected with negative control (NC), miR-212 mimic and miR-212 inhibitor. The scale bar in E indicates 20 μm and n = 5 and 8 in C and D, respectively.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4305303&req=5

pone.0116784.g007: Transfection of miR-132/212 mimics and inhibitors affect MeCP2 expression and cell number in vitro.An initial dosing experiment performed in duplicate using human detrusor smooth muscle cells from one individual shows a reciprocal effect of miR-132 mimic and inhibitor on MeCP2 expression (A). The effect of 100 nM miR-132 mimic was confirmed using cells from three individuals (B). Panel C shows effect of miR-212 mimic and inhibitor (100 nM of each) on cell viability using the MTT assay. Panel D shows the effect of miR-212 mimic and inhibitor on cell number and panel E shows phase contrast light microscopic images of cells transfected with negative control (NC), miR-212 mimic and miR-212 inhibitor. The scale bar in E indicates 20 μm and n = 5 and 8 in C and D, respectively.
Mentions: The lack of support for a role of miR-132/212 in regulation of Ache in the bladder led us to consider alternative roles. Using cultured human detrusor myocytes we transfected miR-132/212 mimics and antimirs. In a dosing experiment we found reciprocal changes in expression of the transcriptional regulator MeCP2 following transfection of miR-132 mimic and antimir (Fig. 7A). A similar trend was seen for miR-212 (not shown). The effect of 100 nM miR-132 mimic on MeCP2 expression was confirmed using cells from three different patients (Fig. 7B). We next examined effects on cell viability and cell number. MiR-212 mimic was found to reduce both viability and cell number (Fig. 7C and D), whereas the inhibitor increased cell number (Fig. 7D). These effects were apparent in bright field images of cells after transfection of miR-212 mimic and inhibitor compared to negative control (NC, Fig. 7E). Signs of apoptosis, such as cell membrane blebbing and cell shrinkage, were not apparent (Fig. 7E). MiR-132 inhibitor increased cell viability (+6.7±1.36%, p<0.01 vs. negative control), while miR-132 mimic was without effect (−0.7±1.6%, p = 0.75).

Bottom Line: The microRNAs (miRNAs) miR-132 and miR-212 have been found to regulate synaptic plasticity and cholinergic signaling and recent work has demonstrated roles outside of the CNS, including in smooth muscle.Moreover, we found that serum stimulation and protein kinase C activation induced miR-132/212 in human detrusor cells.Importantly, transfection of antimirs and mimics in cultured detrusor cells increased and decreased, respectively, the number of cells and led to changes in MeCP2 expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Medical Science, Lund University, Lund, Sweden.

ABSTRACT
The microRNAs (miRNAs) miR-132 and miR-212 have been found to regulate synaptic plasticity and cholinergic signaling and recent work has demonstrated roles outside of the CNS, including in smooth muscle. Here, we examined if miR-132 and miR-212 are induced in the urinary bladder following outlet obstruction and whether this correlates with effects on gene expression and cell growth. Three to seven-fold induction of miR-132/212 was found at 10 days of obstruction and this was selective for the detrusor layer. We cross-referenced putative binding sites in the miR-132/212 promoter with transcription factors that were predicted to be active in the obstruction model. This suggested involvement of Creb and Ahr in miR-132/212 induction. Creb phosphorylation (S-133) was not increased, but the number of Ahr positive nuclei increased. Moreover, we found that serum stimulation and protein kinase C activation induced miR-132/212 in human detrusor cells. To identify miR-132/212 targets, we correlated the mRNA levels of validated targets with the miRNA levels. Significant correlations between miR-132/212 and MeCP2, Ep300, Pnkd and Jarid1a were observed, and the protein levels of MeCP2, Pnkd and Ache were reduced after obstruction. Reduction of Ache however closely matched a 90% reduction of synapse density arguing that its repression was unrelated to miR-132/212 induction. Importantly, transfection of antimirs and mimics in cultured detrusor cells increased and decreased, respectively, the number of cells and led to changes in MeCP2 expression. In all, these findings show that obstruction of the urethra increases miR-132 and miR-212 in the detrusor and suggests that this influences gene expression and limits cell growth.

Show MeSH
Related in: MedlinePlus