Limits...
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: Moreover, we found that serum stimulation and protein kinase C activation induced miR-132/212 in human detrusor cells.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.

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

Bioinformatics analysis points to the involvement of Ahr in miR-132/212 induction following outlet obstruction.Transcription factor binding site (TFBS) analysis identified significant enrichment of 93 transcription factor binding motifs in promoters of differentially expressed genes at 10 days of obstruction (blue circle in A). These were cross-referenced against putative transcription factor binding sites in the miR-132/212 promoter (red circle in A), yielding a list of putative mediators of miR-132/212 induction in outlet obstruction (B). Hits with low expression levels and those with raw p-values exceeding 0.01 are shown in thin lettering. Panel C shows phosphorylation of CREB (S133) in bladders from sham-operated rats and following various time of obstruction. Panel D shows immunofluorescence labeling (green) of the dioxin receptor Ahr in bladders from sham-operated and obstructed rats. Dotted lines indicate the outer surface of the urinary bladder. The scale bar to the left applies to all images and represents 100 μm. Panels E through G show miR-132 expression in human bladder smooth muscle cells stimulated with vehicle (control) and various pharmacological substances in vitro. TCDD: 2,3,7,8-tetrachlorodibenzo-p-dioxin; FCS: fetal calf serum; PMA: Phorbol 12-myristate 13-acetate. Further details are given in Materials and Methods.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4305303&req=5

pone.0116784.g003: Bioinformatics analysis points to the involvement of Ahr in miR-132/212 induction following outlet obstruction.Transcription factor binding site (TFBS) analysis identified significant enrichment of 93 transcription factor binding motifs in promoters of differentially expressed genes at 10 days of obstruction (blue circle in A). These were cross-referenced against putative transcription factor binding sites in the miR-132/212 promoter (red circle in A), yielding a list of putative mediators of miR-132/212 induction in outlet obstruction (B). Hits with low expression levels and those with raw p-values exceeding 0.01 are shown in thin lettering. Panel C shows phosphorylation of CREB (S133) in bladders from sham-operated rats and following various time of obstruction. Panel D shows immunofluorescence labeling (green) of the dioxin receptor Ahr in bladders from sham-operated and obstructed rats. Dotted lines indicate the outer surface of the urinary bladder. The scale bar to the left applies to all images and represents 100 μm. Panels E through G show miR-132 expression in human bladder smooth muscle cells stimulated with vehicle (control) and various pharmacological substances in vitro. TCDD: 2,3,7,8-tetrachlorodibenzo-p-dioxin; FCS: fetal calf serum; PMA: Phorbol 12-myristate 13-acetate. Further details are given in Materials and Methods.

Mentions: Transcription factor binding site (TFBS) analysis is an indirect method to predict involvement of transcription factors using gene lists from microarray experiments. TFBS analysis at 10 days of obstruction (vs. sham) supported involvement of 93 (85+8, Fig. 3A, blue circle) transcription factors in the expression changes resulting from obstruction. We cross-referenced these against putative binding sites in the miR-132/212 promoter (Fig. 3A red circle). Eight transcription factors were represented in both circles of the Venn diagram (Fig. 3A). This list (Fig. 3B) was further shortened by removing transcription factors with expression levels equaling the negative controls in sham-operated and obstructed bladders. The top remaining candidates were the dioxin receptor (Ahr), Early growth response 1 and 2 (Egr1/2), CREB (Creb1) and ATF6α (Atf6).


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)

Bioinformatics analysis points to the involvement of Ahr in miR-132/212 induction following outlet obstruction.Transcription factor binding site (TFBS) analysis identified significant enrichment of 93 transcription factor binding motifs in promoters of differentially expressed genes at 10 days of obstruction (blue circle in A). These were cross-referenced against putative transcription factor binding sites in the miR-132/212 promoter (red circle in A), yielding a list of putative mediators of miR-132/212 induction in outlet obstruction (B). Hits with low expression levels and those with raw p-values exceeding 0.01 are shown in thin lettering. Panel C shows phosphorylation of CREB (S133) in bladders from sham-operated rats and following various time of obstruction. Panel D shows immunofluorescence labeling (green) of the dioxin receptor Ahr in bladders from sham-operated and obstructed rats. Dotted lines indicate the outer surface of the urinary bladder. The scale bar to the left applies to all images and represents 100 μm. Panels E through G show miR-132 expression in human bladder smooth muscle cells stimulated with vehicle (control) and various pharmacological substances in vitro. TCDD: 2,3,7,8-tetrachlorodibenzo-p-dioxin; FCS: fetal calf serum; PMA: Phorbol 12-myristate 13-acetate. Further details are given in Materials and Methods.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0116784.g003: Bioinformatics analysis points to the involvement of Ahr in miR-132/212 induction following outlet obstruction.Transcription factor binding site (TFBS) analysis identified significant enrichment of 93 transcription factor binding motifs in promoters of differentially expressed genes at 10 days of obstruction (blue circle in A). These were cross-referenced against putative transcription factor binding sites in the miR-132/212 promoter (red circle in A), yielding a list of putative mediators of miR-132/212 induction in outlet obstruction (B). Hits with low expression levels and those with raw p-values exceeding 0.01 are shown in thin lettering. Panel C shows phosphorylation of CREB (S133) in bladders from sham-operated rats and following various time of obstruction. Panel D shows immunofluorescence labeling (green) of the dioxin receptor Ahr in bladders from sham-operated and obstructed rats. Dotted lines indicate the outer surface of the urinary bladder. The scale bar to the left applies to all images and represents 100 μm. Panels E through G show miR-132 expression in human bladder smooth muscle cells stimulated with vehicle (control) and various pharmacological substances in vitro. TCDD: 2,3,7,8-tetrachlorodibenzo-p-dioxin; FCS: fetal calf serum; PMA: Phorbol 12-myristate 13-acetate. Further details are given in Materials and Methods.
Mentions: Transcription factor binding site (TFBS) analysis is an indirect method to predict involvement of transcription factors using gene lists from microarray experiments. TFBS analysis at 10 days of obstruction (vs. sham) supported involvement of 93 (85+8, Fig. 3A, blue circle) transcription factors in the expression changes resulting from obstruction. We cross-referenced these against putative binding sites in the miR-132/212 promoter (Fig. 3A red circle). Eight transcription factors were represented in both circles of the Venn diagram (Fig. 3A). This list (Fig. 3B) was further shortened by removing transcription factors with expression levels equaling the negative controls in sham-operated and obstructed bladders. The top remaining candidates were the dioxin receptor (Ahr), Early growth response 1 and 2 (Egr1/2), CREB (Creb1) and ATF6α (Atf6).

Bottom Line: Moreover, we found that serum stimulation and protein kinase C activation induced miR-132/212 in human detrusor cells.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.

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