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Transcriptome analysis using next generation sequencing reveals molecular signatures of diabetic retinopathy and efficacy of candidate drugs.

Kandpal RP, Rajasimha HK, Brooks MJ, Nellissery J, Wan J, Qian J, Kern TS, Swaroop A - Mol. Vis. (2012)

Bottom Line: These two therapies also showed dissimilar regulation of some subsets of transcripts that included alternatively spliced versions of arrestin, neutral sphingomyelinase activation associated factor (Nsmaf), SH3-domain GRB2-like interacting protein 1 (Sgip1), and axin.Diabetes alters many transcripts in the retina, and two therapies that inhibit the vascular pathology similarly inhibit a portion of these changes, pointing to possible molecular mechanisms for their beneficial effects.Our studies clearly demonstrate RNA-seq as a comprehensive strategy for identifying disease-specific transcripts, and for determining comparative profiles of molecular changes mediated by candidate drugs.

View Article: PubMed Central - PubMed

Affiliation: Neurobiology-Neurodegeneration & Repair Laboratory, National Eye Institute, National Institutes of Health, Bethesda, MD 20892, USA. rkandpal@westernu.edu

ABSTRACT

Purpose: To define gene expression changes associated with diabetic retinopathy in a mouse model using next generation sequencing, and to utilize transcriptome signatures to assess molecular pathways by which pharmacological agents inhibit diabetic retinopathy.

Methods: We applied a high throughput RNA sequencing (RNA-seq) strategy using Illumina GAIIx to characterize the entire retinal transcriptome from nondiabetic and from streptozotocin-treated mice 32 weeks after induction of diabetes. Some of the diabetic mice were treated with inhibitors of receptor for advanced glycation endproducts (RAGE) and p38 mitogen activated protein (MAP) kinase, which have previously been shown to inhibit diabetic retinopathy in rodent models. The transcripts and alternatively spliced variants were determined in all experimental groups.

Results: Next generation sequencing-based RNA-seq profiles provided comprehensive signatures of transcripts that are altered in early stages of diabetic retinopathy. These transcripts encoded proteins involved in distinct yet physiologically relevant disease-associated pathways such as inflammation, microvasculature formation, apoptosis, glucose metabolism, Wnt signaling, xenobiotic metabolism, and photoreceptor biology. Significant upregulation of crystallin transcripts was observed in diabetic animals, and the diabetes-induced upregulation of these transcripts was inhibited in diabetic animals treated with inhibitors of either RAGE or p38 MAP kinase. These two therapies also showed dissimilar regulation of some subsets of transcripts that included alternatively spliced versions of arrestin, neutral sphingomyelinase activation associated factor (Nsmaf), SH3-domain GRB2-like interacting protein 1 (Sgip1), and axin.

Conclusions: Diabetes alters many transcripts in the retina, and two therapies that inhibit the vascular pathology similarly inhibit a portion of these changes, pointing to possible molecular mechanisms for their beneficial effects. These therapies also changed the abundance of various alternatively spliced versions of signaling transcripts, suggesting a possible role of alternative splicing in disease etiology. Our studies clearly demonstrate RNA-seq as a comprehensive strategy for identifying disease-specific transcripts, and for determining comparative profiles of molecular changes mediated by candidate drugs.

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Differentially expressed gene sets were obtained by employing either significance of microarray analysis algorithm or ANOVA method. The gene sets were applied as described in Methods section and the genes unique and common in the two sets are indicated.
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f1: Differentially expressed gene sets were obtained by employing either significance of microarray analysis algorithm or ANOVA method. The gene sets were applied as described in Methods section and the genes unique and common in the two sets are indicated.

Mentions: We analyzed the sequence data using ELAND-SAM and BWA-ANOVA methods, and compared the results to confirm method-specific changes in transcript abundance in the various groups of animals. Differential expression in diabetic and normal animals revealed the top 100 transcripts shortlisted by the two methods, as shown in Table 2 (Appendix 1). Of these 100 transcripts, 81 are present in both data sets (Figure 1) including a variety of crystallin genes. However, UDP-glucuronosyl transferase transcripts were detected only by the ELAND-SAM method. A significant difference was also noted in the fold-changes indicated by the two methods. These differences can be attributed to differences in the alignment algorithms, filtering criteria, and calculation of differential gene expression between the two methods, and demonstrate that altered abundance of transcripts can be detected by either one of these two methods.


Transcriptome analysis using next generation sequencing reveals molecular signatures of diabetic retinopathy and efficacy of candidate drugs.

Kandpal RP, Rajasimha HK, Brooks MJ, Nellissery J, Wan J, Qian J, Kern TS, Swaroop A - Mol. Vis. (2012)

Differentially expressed gene sets were obtained by employing either significance of microarray analysis algorithm or ANOVA method. The gene sets were applied as described in Methods section and the genes unique and common in the two sets are indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3351417&req=5

f1: Differentially expressed gene sets were obtained by employing either significance of microarray analysis algorithm or ANOVA method. The gene sets were applied as described in Methods section and the genes unique and common in the two sets are indicated.
Mentions: We analyzed the sequence data using ELAND-SAM and BWA-ANOVA methods, and compared the results to confirm method-specific changes in transcript abundance in the various groups of animals. Differential expression in diabetic and normal animals revealed the top 100 transcripts shortlisted by the two methods, as shown in Table 2 (Appendix 1). Of these 100 transcripts, 81 are present in both data sets (Figure 1) including a variety of crystallin genes. However, UDP-glucuronosyl transferase transcripts were detected only by the ELAND-SAM method. A significant difference was also noted in the fold-changes indicated by the two methods. These differences can be attributed to differences in the alignment algorithms, filtering criteria, and calculation of differential gene expression between the two methods, and demonstrate that altered abundance of transcripts can be detected by either one of these two methods.

Bottom Line: These two therapies also showed dissimilar regulation of some subsets of transcripts that included alternatively spliced versions of arrestin, neutral sphingomyelinase activation associated factor (Nsmaf), SH3-domain GRB2-like interacting protein 1 (Sgip1), and axin.Diabetes alters many transcripts in the retina, and two therapies that inhibit the vascular pathology similarly inhibit a portion of these changes, pointing to possible molecular mechanisms for their beneficial effects.Our studies clearly demonstrate RNA-seq as a comprehensive strategy for identifying disease-specific transcripts, and for determining comparative profiles of molecular changes mediated by candidate drugs.

View Article: PubMed Central - PubMed

Affiliation: Neurobiology-Neurodegeneration & Repair Laboratory, National Eye Institute, National Institutes of Health, Bethesda, MD 20892, USA. rkandpal@westernu.edu

ABSTRACT

Purpose: To define gene expression changes associated with diabetic retinopathy in a mouse model using next generation sequencing, and to utilize transcriptome signatures to assess molecular pathways by which pharmacological agents inhibit diabetic retinopathy.

Methods: We applied a high throughput RNA sequencing (RNA-seq) strategy using Illumina GAIIx to characterize the entire retinal transcriptome from nondiabetic and from streptozotocin-treated mice 32 weeks after induction of diabetes. Some of the diabetic mice were treated with inhibitors of receptor for advanced glycation endproducts (RAGE) and p38 mitogen activated protein (MAP) kinase, which have previously been shown to inhibit diabetic retinopathy in rodent models. The transcripts and alternatively spliced variants were determined in all experimental groups.

Results: Next generation sequencing-based RNA-seq profiles provided comprehensive signatures of transcripts that are altered in early stages of diabetic retinopathy. These transcripts encoded proteins involved in distinct yet physiologically relevant disease-associated pathways such as inflammation, microvasculature formation, apoptosis, glucose metabolism, Wnt signaling, xenobiotic metabolism, and photoreceptor biology. Significant upregulation of crystallin transcripts was observed in diabetic animals, and the diabetes-induced upregulation of these transcripts was inhibited in diabetic animals treated with inhibitors of either RAGE or p38 MAP kinase. These two therapies also showed dissimilar regulation of some subsets of transcripts that included alternatively spliced versions of arrestin, neutral sphingomyelinase activation associated factor (Nsmaf), SH3-domain GRB2-like interacting protein 1 (Sgip1), and axin.

Conclusions: Diabetes alters many transcripts in the retina, and two therapies that inhibit the vascular pathology similarly inhibit a portion of these changes, pointing to possible molecular mechanisms for their beneficial effects. These therapies also changed the abundance of various alternatively spliced versions of signaling transcripts, suggesting a possible role of alternative splicing in disease etiology. Our studies clearly demonstrate RNA-seq as a comprehensive strategy for identifying disease-specific transcripts, and for determining comparative profiles of molecular changes mediated by candidate drugs.

Show MeSH
Related in: MedlinePlus