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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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Related in: MedlinePlus

TaqMan real-time quantitative polymerase chain reaction (qPCR) validation was performed for selected transcripts. The transcripts were amplified as described in Methods section. Three technical replicates were performed for three biologic replicate samples of RNA isolated from nondiabetic animals, diabetic animals and diabetic animals treated with either the inhibitor of receptor for advanced glycation endproducts (RAGE) indicated as RI, or the inhibitor of p38 mitogen activated protein kinase (MAPK) designated as p38. The bars (left to right) represent samples corresponding to diabetic animals, diabetic animals treated with RAGE inhibitor, and diabetic animals treated with p38 MAPK inhibitor, respectively. Fold increase was calculated with respect to nondiabetic controls.
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f2: TaqMan real-time quantitative polymerase chain reaction (qPCR) validation was performed for selected transcripts. The transcripts were amplified as described in Methods section. Three technical replicates were performed for three biologic replicate samples of RNA isolated from nondiabetic animals, diabetic animals and diabetic animals treated with either the inhibitor of receptor for advanced glycation endproducts (RAGE) indicated as RI, or the inhibitor of p38 mitogen activated protein kinase (MAPK) designated as p38. The bars (left to right) represent samples corresponding to diabetic animals, diabetic animals treated with RAGE inhibitor, and diabetic animals treated with p38 MAPK inhibitor, respectively. Fold increase was calculated with respect to nondiabetic controls.

Mentions: Some representative transcripts were chosen for validation based on the magnitude of the changes in their abundance and their involvement in various pathways. We performed Taqman real-time PCR to validate 11 transcripts, including Cryga-crystallin gamma A, Crygb-crystallin gamma B, Dct-dopachrome tautomerase, Egr-early growth response, FABP-fatty acid binding protein, Lgsn-lengsin, Lim2-lens intrinsic membrane protein, Nr2c2ap-nuclear receptor 2C2-associated protein, retinal pigment epithelium 65 Kd protein (Rpe65), Sfrp1-secreted frizzled-related protein, and wingless type MMTV integration site, member 7b (Wnt7b). The level of each of these transcripts corresponded to the pattern revealed by RNA-seq, and indicated significant changes in the levels of these transcripts between diabetic and normal mice (Figure 2). However, the magnitude of change observed by RT–PCR was not identical to the results of RNA-seq. The non-correspondence of magnitude reflects the differences in sensitivities of the RNA-seq and Taqman assays, which are two inherently different strategies.


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)

TaqMan real-time quantitative polymerase chain reaction (qPCR) validation was performed for selected transcripts. The transcripts were amplified as described in Methods section. Three technical replicates were performed for three biologic replicate samples of RNA isolated from nondiabetic animals, diabetic animals and diabetic animals treated with either the inhibitor of receptor for advanced glycation endproducts (RAGE) indicated as RI, or the inhibitor of p38 mitogen activated protein kinase (MAPK) designated as p38. The bars (left to right) represent samples corresponding to diabetic animals, diabetic animals treated with RAGE inhibitor, and diabetic animals treated with p38 MAPK inhibitor, respectively. Fold increase was calculated with respect to nondiabetic controls.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: TaqMan real-time quantitative polymerase chain reaction (qPCR) validation was performed for selected transcripts. The transcripts were amplified as described in Methods section. Three technical replicates were performed for three biologic replicate samples of RNA isolated from nondiabetic animals, diabetic animals and diabetic animals treated with either the inhibitor of receptor for advanced glycation endproducts (RAGE) indicated as RI, or the inhibitor of p38 mitogen activated protein kinase (MAPK) designated as p38. The bars (left to right) represent samples corresponding to diabetic animals, diabetic animals treated with RAGE inhibitor, and diabetic animals treated with p38 MAPK inhibitor, respectively. Fold increase was calculated with respect to nondiabetic controls.
Mentions: Some representative transcripts were chosen for validation based on the magnitude of the changes in their abundance and their involvement in various pathways. We performed Taqman real-time PCR to validate 11 transcripts, including Cryga-crystallin gamma A, Crygb-crystallin gamma B, Dct-dopachrome tautomerase, Egr-early growth response, FABP-fatty acid binding protein, Lgsn-lengsin, Lim2-lens intrinsic membrane protein, Nr2c2ap-nuclear receptor 2C2-associated protein, retinal pigment epithelium 65 Kd protein (Rpe65), Sfrp1-secreted frizzled-related protein, and wingless type MMTV integration site, member 7b (Wnt7b). The level of each of these transcripts corresponded to the pattern revealed by RNA-seq, and indicated significant changes in the levels of these transcripts between diabetic and normal mice (Figure 2). However, the magnitude of change observed by RT–PCR was not identical to the results of RNA-seq. The non-correspondence of magnitude reflects the differences in sensitivities of the RNA-seq and Taqman assays, which are two inherently different strategies.

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