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Prediction of microRNAs affecting mRNA expression during retinal development.

Arora A, Guduric-Fuchs J, Harwood L, Dellett M, Cogliati T, Simpson DA - BMC Dev. Biol. (2010)

Bottom Line: The expression levels of miRNAs correlated with the significance of their predicted effects upon mRNA expression.Over-expression of three miRNAs with significant predicted effects upon global mRNA levels resulted in a decrease in mRNA expression of five out of six individual predicted target genes assayed.Conversely, manipulation of their expression could potentially be used as a therapeutic tool in the future.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Vision and Vascular Science, Queen's University Belfast, Ophthalmic Research Centre, Institute of Clinical Science, Royal Victoria Hospital, Belfast BT12 6BA, UK.

ABSTRACT

Background: MicroRNAs (miRNAs) are small RNA molecules (~22 nucleotides) which have been shown to play an important role both in development and in maintenance of adult tissue. Conditional inactivation of miRNAs in the eye causes loss of visual function and progressive retinal degeneration. In addition to inhibiting translation, miRNAs can mediate degradation of targeted mRNAs. We have previously shown that candidate miRNAs affecting transcript levels in a tissue can be deduced from mRNA microarray expression profiles. The purpose of this study was to predict miRNAs which affect mRNA levels in developing and adult retinal tissue and to confirm their expression.

Results: Microarray expression data from ciliary epithelial retinal stem cells (CE-RSCs), developing and adult mouse retina were generated or downloaded from public repositories. Analysis of gene expression profiles detected the effects of multiple miRNAs in CE-RSCs and retina. The expression of 20 selected miRNAs was confirmed by RT-PCR and the cellular distribution of representative candidates analyzed by in situ hybridization. The expression levels of miRNAs correlated with the significance of their predicted effects upon mRNA expression. Highly expressed miRNAs included miR-124, miR-125a, miR-125b, miR-204 and miR-9. Over-expression of three miRNAs with significant predicted effects upon global mRNA levels resulted in a decrease in mRNA expression of five out of six individual predicted target genes assayed.

Conclusions: This study has detected the effect of miRNAs upon mRNA expression in immature and adult retinal tissue and cells. The validity of these observations is supported by the experimental confirmation of candidate miRNA expression and the regulation of predicted target genes following miRNA over-expression. Identified miRNAs are likely to be important in retinal development and function. Misregulation of these miRNAs might contribute to retinal degeneration and disease. Conversely, manipulation of their expression could potentially be used as a therapeutic tool in the future.

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Cellular expression of selected miRNAs. ISH was performed on porcine CE-RSC neurospheres, and P4 and adult mouse retinas. Some neurosphere cells are naturally pigmented (dark brown). A: No positive signal was detected for miR-124 in CE-RSC neurospheres. B: Expression of miR-124 at P4 corresponded to the location of amacrine (arrows) and ganglion cells (arrowheads). C: In the adult retina, miR-124 was expressed in all layers with the highest intensity in the photoreceptor inner segments (arrows) and cells in the INL (arrowheads). D: miR-125b-5p was detected in the RSC neurospheres (purple staining depicted with arrowheads). E: In the P4 retina strong expression of miR-125b-5p was found in the inner portion of the NBL (arrows) and GCL (arrowheads). F: In the adult retina expression of miR-125b was detected in the ONL (arrows) and INL (arrowheads). G: Weak hybridisation signal for miR-34a was observed in CE-RSC neurosphere cells (arrowheads). H: P4 retina displayed week miR-34a signal throughout NBL (arrows) and ganglion (arrowheads) I: In the adult retina the strongest signal for miR-34a was observed in the INL (arrows) and in some cells in the GCL (arrowheads). J: Strong hybridisation signal was detected for miR-128 in the CE-RSC neurospheres (arrowheads). K: In the P4 retina a hybridisation signal for miR-128 was observed in the inner portion of the NBL (arrows) and in some cells of the GCL (arrowheads). L: In the adult retina strongest miR-128 hybridisation signal was detected in the INL (arrowheads), the outer portion of the outer plexiform layer and photoreceptor inner segments (arrows). ISH with scrambled negative control gave no signal in CE-RSCs (M), P4 (N), or adult mouse retina (O). NBL-neuroblast layer, GCL-ganglion cell layer, INL-inner nuclear layer, ONL-outer nuclear layer. Scale bars 50 μm.
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Figure 5: Cellular expression of selected miRNAs. ISH was performed on porcine CE-RSC neurospheres, and P4 and adult mouse retinas. Some neurosphere cells are naturally pigmented (dark brown). A: No positive signal was detected for miR-124 in CE-RSC neurospheres. B: Expression of miR-124 at P4 corresponded to the location of amacrine (arrows) and ganglion cells (arrowheads). C: In the adult retina, miR-124 was expressed in all layers with the highest intensity in the photoreceptor inner segments (arrows) and cells in the INL (arrowheads). D: miR-125b-5p was detected in the RSC neurospheres (purple staining depicted with arrowheads). E: In the P4 retina strong expression of miR-125b-5p was found in the inner portion of the NBL (arrows) and GCL (arrowheads). F: In the adult retina expression of miR-125b was detected in the ONL (arrows) and INL (arrowheads). G: Weak hybridisation signal for miR-34a was observed in CE-RSC neurosphere cells (arrowheads). H: P4 retina displayed week miR-34a signal throughout NBL (arrows) and ganglion (arrowheads) I: In the adult retina the strongest signal for miR-34a was observed in the INL (arrows) and in some cells in the GCL (arrowheads). J: Strong hybridisation signal was detected for miR-128 in the CE-RSC neurospheres (arrowheads). K: In the P4 retina a hybridisation signal for miR-128 was observed in the inner portion of the NBL (arrows) and in some cells of the GCL (arrowheads). L: In the adult retina strongest miR-128 hybridisation signal was detected in the INL (arrowheads), the outer portion of the outer plexiform layer and photoreceptor inner segments (arrows). ISH with scrambled negative control gave no signal in CE-RSCs (M), P4 (N), or adult mouse retina (O). NBL-neuroblast layer, GCL-ganglion cell layer, INL-inner nuclear layer, ONL-outer nuclear layer. Scale bars 50 μm.

Mentions: In situ hybridization (ISH) was performed to localise expression of selected miRNAs (Figure 5). Expression in the adult murine retina of the well-characterised neural miRNA miR-124 concurred with published reports [9]. In P4 retina, miR-124 positive signal was mainly colocalised with ganglion and amacrine cells. Expression of miR-34a, which has not previously been described in the retina, was detected in the porcine CE-RSC neurospheres and throughout the mouse P4 retina. In the adult mouse retina the strongest signal was observed in the inner nuclear layer (INL) and in some cells in the ganglion cell layer (GCL). miR-128 was expressed in all samples, notably in CE-RSC neurospheres and the inner plexiform layer of the retina at P4. miR-125b was also expressed at all stages with strong signal in the inner plexiform layer at P4. In the adult retina miR-125b was expressed in the inner and outer nuclear layers. No expression was observed for miR-122 and miR-378 (data not shown).


Prediction of microRNAs affecting mRNA expression during retinal development.

Arora A, Guduric-Fuchs J, Harwood L, Dellett M, Cogliati T, Simpson DA - BMC Dev. Biol. (2010)

Cellular expression of selected miRNAs. ISH was performed on porcine CE-RSC neurospheres, and P4 and adult mouse retinas. Some neurosphere cells are naturally pigmented (dark brown). A: No positive signal was detected for miR-124 in CE-RSC neurospheres. B: Expression of miR-124 at P4 corresponded to the location of amacrine (arrows) and ganglion cells (arrowheads). C: In the adult retina, miR-124 was expressed in all layers with the highest intensity in the photoreceptor inner segments (arrows) and cells in the INL (arrowheads). D: miR-125b-5p was detected in the RSC neurospheres (purple staining depicted with arrowheads). E: In the P4 retina strong expression of miR-125b-5p was found in the inner portion of the NBL (arrows) and GCL (arrowheads). F: In the adult retina expression of miR-125b was detected in the ONL (arrows) and INL (arrowheads). G: Weak hybridisation signal for miR-34a was observed in CE-RSC neurosphere cells (arrowheads). H: P4 retina displayed week miR-34a signal throughout NBL (arrows) and ganglion (arrowheads) I: In the adult retina the strongest signal for miR-34a was observed in the INL (arrows) and in some cells in the GCL (arrowheads). J: Strong hybridisation signal was detected for miR-128 in the CE-RSC neurospheres (arrowheads). K: In the P4 retina a hybridisation signal for miR-128 was observed in the inner portion of the NBL (arrows) and in some cells of the GCL (arrowheads). L: In the adult retina strongest miR-128 hybridisation signal was detected in the INL (arrowheads), the outer portion of the outer plexiform layer and photoreceptor inner segments (arrows). ISH with scrambled negative control gave no signal in CE-RSCs (M), P4 (N), or adult mouse retina (O). NBL-neuroblast layer, GCL-ganglion cell layer, INL-inner nuclear layer, ONL-outer nuclear layer. Scale bars 50 μm.
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Figure 5: Cellular expression of selected miRNAs. ISH was performed on porcine CE-RSC neurospheres, and P4 and adult mouse retinas. Some neurosphere cells are naturally pigmented (dark brown). A: No positive signal was detected for miR-124 in CE-RSC neurospheres. B: Expression of miR-124 at P4 corresponded to the location of amacrine (arrows) and ganglion cells (arrowheads). C: In the adult retina, miR-124 was expressed in all layers with the highest intensity in the photoreceptor inner segments (arrows) and cells in the INL (arrowheads). D: miR-125b-5p was detected in the RSC neurospheres (purple staining depicted with arrowheads). E: In the P4 retina strong expression of miR-125b-5p was found in the inner portion of the NBL (arrows) and GCL (arrowheads). F: In the adult retina expression of miR-125b was detected in the ONL (arrows) and INL (arrowheads). G: Weak hybridisation signal for miR-34a was observed in CE-RSC neurosphere cells (arrowheads). H: P4 retina displayed week miR-34a signal throughout NBL (arrows) and ganglion (arrowheads) I: In the adult retina the strongest signal for miR-34a was observed in the INL (arrows) and in some cells in the GCL (arrowheads). J: Strong hybridisation signal was detected for miR-128 in the CE-RSC neurospheres (arrowheads). K: In the P4 retina a hybridisation signal for miR-128 was observed in the inner portion of the NBL (arrows) and in some cells of the GCL (arrowheads). L: In the adult retina strongest miR-128 hybridisation signal was detected in the INL (arrowheads), the outer portion of the outer plexiform layer and photoreceptor inner segments (arrows). ISH with scrambled negative control gave no signal in CE-RSCs (M), P4 (N), or adult mouse retina (O). NBL-neuroblast layer, GCL-ganglion cell layer, INL-inner nuclear layer, ONL-outer nuclear layer. Scale bars 50 μm.
Mentions: In situ hybridization (ISH) was performed to localise expression of selected miRNAs (Figure 5). Expression in the adult murine retina of the well-characterised neural miRNA miR-124 concurred with published reports [9]. In P4 retina, miR-124 positive signal was mainly colocalised with ganglion and amacrine cells. Expression of miR-34a, which has not previously been described in the retina, was detected in the porcine CE-RSC neurospheres and throughout the mouse P4 retina. In the adult mouse retina the strongest signal was observed in the inner nuclear layer (INL) and in some cells in the ganglion cell layer (GCL). miR-128 was expressed in all samples, notably in CE-RSC neurospheres and the inner plexiform layer of the retina at P4. miR-125b was also expressed at all stages with strong signal in the inner plexiform layer at P4. In the adult retina miR-125b was expressed in the inner and outer nuclear layers. No expression was observed for miR-122 and miR-378 (data not shown).

Bottom Line: The expression levels of miRNAs correlated with the significance of their predicted effects upon mRNA expression.Over-expression of three miRNAs with significant predicted effects upon global mRNA levels resulted in a decrease in mRNA expression of five out of six individual predicted target genes assayed.Conversely, manipulation of their expression could potentially be used as a therapeutic tool in the future.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Vision and Vascular Science, Queen's University Belfast, Ophthalmic Research Centre, Institute of Clinical Science, Royal Victoria Hospital, Belfast BT12 6BA, UK.

ABSTRACT

Background: MicroRNAs (miRNAs) are small RNA molecules (~22 nucleotides) which have been shown to play an important role both in development and in maintenance of adult tissue. Conditional inactivation of miRNAs in the eye causes loss of visual function and progressive retinal degeneration. In addition to inhibiting translation, miRNAs can mediate degradation of targeted mRNAs. We have previously shown that candidate miRNAs affecting transcript levels in a tissue can be deduced from mRNA microarray expression profiles. The purpose of this study was to predict miRNAs which affect mRNA levels in developing and adult retinal tissue and to confirm their expression.

Results: Microarray expression data from ciliary epithelial retinal stem cells (CE-RSCs), developing and adult mouse retina were generated or downloaded from public repositories. Analysis of gene expression profiles detected the effects of multiple miRNAs in CE-RSCs and retina. The expression of 20 selected miRNAs was confirmed by RT-PCR and the cellular distribution of representative candidates analyzed by in situ hybridization. The expression levels of miRNAs correlated with the significance of their predicted effects upon mRNA expression. Highly expressed miRNAs included miR-124, miR-125a, miR-125b, miR-204 and miR-9. Over-expression of three miRNAs with significant predicted effects upon global mRNA levels resulted in a decrease in mRNA expression of five out of six individual predicted target genes assayed.

Conclusions: This study has detected the effect of miRNAs upon mRNA expression in immature and adult retinal tissue and cells. The validity of these observations is supported by the experimental confirmation of candidate miRNA expression and the regulation of predicted target genes following miRNA over-expression. Identified miRNAs are likely to be important in retinal development and function. Misregulation of these miRNAs might contribute to retinal degeneration and disease. Conversely, manipulation of their expression could potentially be used as a therapeutic tool in the future.

Show MeSH
Related in: MedlinePlus