Limits...
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.

Show MeSH

Related in: MedlinePlus

Comparision between the predicted effects of miRNAs and their expression as determined by RT-PCR. The estimated copy number (Log(initial template)) of each miRNA in a given sample is plotted against the probability (Wilcoxon ranked sum test) that it is having an effect upon target gene expression. The most highly expressed miRNAs have very significant effects upon predicted target gene expression and the overall correlation between miRNA expression and significance of effect upon target gene expression is significant (p = 4.0E-04; Spearman rank correlation).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2821300&req=5

Figure 4: Comparision between the predicted effects of miRNAs and their expression as determined by RT-PCR. The estimated copy number (Log(initial template)) of each miRNA in a given sample is plotted against the probability (Wilcoxon ranked sum test) that it is having an effect upon target gene expression. The most highly expressed miRNAs have very significant effects upon predicted target gene expression and the overall correlation between miRNA expression and significance of effect upon target gene expression is significant (p = 4.0E-04; Spearman rank correlation).

Mentions: The pattern of relative expression in the three samples considered (porcine CE-RSCs, mouse P4 and adult retina) varied widely amongst miRNAs (Figure 3A). The expression of each individual miRNA corresponded broadly with its predicted effects upon target gene expression (e.g. miR-25 highest at P4 and miR-124 absent in CE-RSCs but present in P4 and adult mouse retina). However, whilst this analysis indicates the sample in which the relative expression of a single miRNA is highest, it gives no indication of the inter-miRNA variation in expression in that sample. It is also important to consider the relative absolute expression of different miRNAs within a single sample when considering the impact of each upon target mRNA expression. Therefore, starting template copy numbers were estimated for each miRNA based on threshold cycle (Ct) and amplification efficiency or by the linear regression efficiency method [31], which were broadly in agreement (Additional file 2: Figure S1). The miRNAs measured in each sample are shown in Figure 3B ranked by their starting template copy number in the RT-PCR assays. Those miRNAs with the most significant predicted effects upon mRNA expression are highly expressed. Notably, miR-125 has amongst the most significant predicted effects upon mRNA expression at all stages (Table 1) and miR-125a and miR-125b are the most highly expressed miRNAs in all samples (Figure 3B). Furthermore, miR-124 is highly expressed in P4 and adult retina in accordance with its predicted effects. To test the expected relationship between predicted effects and miRNA expression, the estimated miRNA copy numbers were plotted against predicted probability (Figure 4). This revealed a significant correlation (Spearman rank correlation, p = 4.0E-04), with the majority of most highly expressed miRNAs having highly significant predicted effects.


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)

Comparision between the predicted effects of miRNAs and their expression as determined by RT-PCR. The estimated copy number (Log(initial template)) of each miRNA in a given sample is plotted against the probability (Wilcoxon ranked sum test) that it is having an effect upon target gene expression. The most highly expressed miRNAs have very significant effects upon predicted target gene expression and the overall correlation between miRNA expression and significance of effect upon target gene expression is significant (p = 4.0E-04; Spearman rank correlation).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2821300&req=5

Figure 4: Comparision between the predicted effects of miRNAs and their expression as determined by RT-PCR. The estimated copy number (Log(initial template)) of each miRNA in a given sample is plotted against the probability (Wilcoxon ranked sum test) that it is having an effect upon target gene expression. The most highly expressed miRNAs have very significant effects upon predicted target gene expression and the overall correlation between miRNA expression and significance of effect upon target gene expression is significant (p = 4.0E-04; Spearman rank correlation).
Mentions: The pattern of relative expression in the three samples considered (porcine CE-RSCs, mouse P4 and adult retina) varied widely amongst miRNAs (Figure 3A). The expression of each individual miRNA corresponded broadly with its predicted effects upon target gene expression (e.g. miR-25 highest at P4 and miR-124 absent in CE-RSCs but present in P4 and adult mouse retina). However, whilst this analysis indicates the sample in which the relative expression of a single miRNA is highest, it gives no indication of the inter-miRNA variation in expression in that sample. It is also important to consider the relative absolute expression of different miRNAs within a single sample when considering the impact of each upon target mRNA expression. Therefore, starting template copy numbers were estimated for each miRNA based on threshold cycle (Ct) and amplification efficiency or by the linear regression efficiency method [31], which were broadly in agreement (Additional file 2: Figure S1). The miRNAs measured in each sample are shown in Figure 3B ranked by their starting template copy number in the RT-PCR assays. Those miRNAs with the most significant predicted effects upon mRNA expression are highly expressed. Notably, miR-125 has amongst the most significant predicted effects upon mRNA expression at all stages (Table 1) and miR-125a and miR-125b are the most highly expressed miRNAs in all samples (Figure 3B). Furthermore, miR-124 is highly expressed in P4 and adult retina in accordance with its predicted effects. To test the expected relationship between predicted effects and miRNA expression, the estimated miRNA copy numbers were plotted against predicted probability (Figure 4). This revealed a significant correlation (Spearman rank correlation, p = 4.0E-04), with the majority of most highly expressed miRNAs having highly significant predicted effects.

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