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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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Partial infravesical outlet obstruction increases expression of miR-132/212 in the urinary bladder.Rats were sham-operated (sham) or subjected to obstruction for 10 days (10d obs) and 6 weeks (6w obs). One group of rats were obstructed for 6 weeks and then re-operated to remove the obstruction. The obstruction-induced hypertrophy was then allowed to regress for another 10 days (de-obs). Bladders were harvested, RNA was isolated and microarrays were run (GEO accession GSE47080). Results in panels A and B are from these microarrays. Panels C and D show confirmation using qRT-PCR at 10 days of obstruction versus sham. *, **, and *** denote p<0.05, p<0.01 and p<0.001 in this and the following figures. N = 6–8 throughout.
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pone.0116784.g001: Partial infravesical outlet obstruction increases expression of miR-132/212 in the urinary bladder.Rats were sham-operated (sham) or subjected to obstruction for 10 days (10d obs) and 6 weeks (6w obs). One group of rats were obstructed for 6 weeks and then re-operated to remove the obstruction. The obstruction-induced hypertrophy was then allowed to regress for another 10 days (de-obs). Bladders were harvested, RNA was isolated and microarrays were run (GEO accession GSE47080). Results in panels A and B are from these microarrays. Panels C and D show confirmation using qRT-PCR at 10 days of obstruction versus sham. *, **, and *** denote p<0.05, p<0.01 and p<0.001 in this and the following figures. N = 6–8 throughout.

Mentions: Microarrays using bladders obstructed for 10 days and at 6 weeks as well as de-obstructed and sham-operated bladders (GEO: GSE47080) demonstrated induction of miR-132 (rno-miR-132-3p) and of miR-212 (rno-miR-212-3p) in bladder outlet obstruction and a return towards control level on de-obstruction (Fig. 1A, B). The corresponding star sequences (5p) were similarly induced (rno-miR-132-5p: 3.2-fold, p = 0.0005; rno-miR-212-5p: 4.0-fold, p = 0.0001). The changes at 10 days were confirmed using qRT-PCR (Fig. 1C, D).


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)

Partial infravesical outlet obstruction increases expression of miR-132/212 in the urinary bladder.Rats were sham-operated (sham) or subjected to obstruction for 10 days (10d obs) and 6 weeks (6w obs). One group of rats were obstructed for 6 weeks and then re-operated to remove the obstruction. The obstruction-induced hypertrophy was then allowed to regress for another 10 days (de-obs). Bladders were harvested, RNA was isolated and microarrays were run (GEO accession GSE47080). Results in panels A and B are from these microarrays. Panels C and D show confirmation using qRT-PCR at 10 days of obstruction versus sham. *, **, and *** denote p<0.05, p<0.01 and p<0.001 in this and the following figures. N = 6–8 throughout.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0116784.g001: Partial infravesical outlet obstruction increases expression of miR-132/212 in the urinary bladder.Rats were sham-operated (sham) or subjected to obstruction for 10 days (10d obs) and 6 weeks (6w obs). One group of rats were obstructed for 6 weeks and then re-operated to remove the obstruction. The obstruction-induced hypertrophy was then allowed to regress for another 10 days (de-obs). Bladders were harvested, RNA was isolated and microarrays were run (GEO accession GSE47080). Results in panels A and B are from these microarrays. Panels C and D show confirmation using qRT-PCR at 10 days of obstruction versus sham. *, **, and *** denote p<0.05, p<0.01 and p<0.001 in this and the following figures. N = 6–8 throughout.
Mentions: Microarrays using bladders obstructed for 10 days and at 6 weeks as well as de-obstructed and sham-operated bladders (GEO: GSE47080) demonstrated induction of miR-132 (rno-miR-132-3p) and of miR-212 (rno-miR-212-3p) in bladder outlet obstruction and a return towards control level on de-obstruction (Fig. 1A, B). The corresponding star sequences (5p) were similarly induced (rno-miR-132-5p: 3.2-fold, p = 0.0005; rno-miR-212-5p: 4.0-fold, p = 0.0001). The changes at 10 days were confirmed using qRT-PCR (Fig. 1C, D).

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