Abstract Title

miR-200b-3p and miR-211-5p downregulate the expression of extracellular matrix and associated proteins in human optic nerve head astrocytes

Presenter Name

Navita Lopez

RAD Assignment Number

504

Abstract

PURPOSE: microRNAs (miRNAs) are a class of small, endogenous non-coding RNAs that epigenetically regulate post-transcriptional gene expression. miRNAs are known to modulate cellular functions such as extracellular matrix (ECM) turnover. There is evidence that dysregulation of miRNA expression has a role in the pathogenesis of fibrotic diseases including glaucoma. Glaucoma is a leading cause of irreversible blindness and is associated with fibrotic changes to the optic nerve head (ONH), the initial site of glaucomatous damage to the retina and optic nerve. Our previous study showed that expression of the profibrotic cytokine TGFβ2 is elevated in the ONH of glaucoma eyes compared to age-matched normal eyes. Currently there is a lack of knowledge regarding the roles of miRNAs in the ONH. The purpose of this study was to determine: (a) differences in the expression of profibrotic and anti-fibrotic miRNAs in normal ONH astrocytes treated with or without TGFβ2 and (b) whether candidate miRNAs (miR-200b-3p and miR-211-5p) regulate the expression of ECM and ECM associated proteins in the ONH.

METHODS: Primary normal human ONH astrocytes (ONA) were grown to 100% confluency. ONA were treated with 5ng/ml TGFβ2 or with control for 24hrs. miRNA qPCR arrays were performed to compare the expression levels of profibrotic and anti-fibrotic miRNAs in normal ONA treated with or without TGFβ2. ONA were transfected with miR-200b-3p and miR-211-5p mimics at 10nM, 5nM, and 1nM concentrations to confirm target predictions based on the TargetScan database. An all stars negative control siRNA was included which will not recognise any mammalian gene.

RESULTS: The miRNA qPCR arrays analysed from normal ONA exposed to TGFβ2 showed that TGFβ2 downregulated the expression of hsa-miR-200b-3p and hsa-miR-211-5p. Transfection of miR-200b-3p mim downregulated fibronectin (FN), gremlin, and tissue transglutaminase II in ONA. Transfection of miR-211-5p downregulated FN and gremlin in ONA .

CONCLUSIONS: Our results suggest that TGFβ2, which is elevated in the glaucomatous ONH, modulates the expression of miRNAs in ONA. These miRNAs target FN, TGM2, and gremlin to modify the ECM in the ONH. Downregulation of anti-fibrotic miRNAs may contribute to fibrosis of the ONH.

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Cell Biology

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Poster

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miR-200b-3p and miR-211-5p downregulate the expression of extracellular matrix and associated proteins in human optic nerve head astrocytes

PURPOSE: microRNAs (miRNAs) are a class of small, endogenous non-coding RNAs that epigenetically regulate post-transcriptional gene expression. miRNAs are known to modulate cellular functions such as extracellular matrix (ECM) turnover. There is evidence that dysregulation of miRNA expression has a role in the pathogenesis of fibrotic diseases including glaucoma. Glaucoma is a leading cause of irreversible blindness and is associated with fibrotic changes to the optic nerve head (ONH), the initial site of glaucomatous damage to the retina and optic nerve. Our previous study showed that expression of the profibrotic cytokine TGFβ2 is elevated in the ONH of glaucoma eyes compared to age-matched normal eyes. Currently there is a lack of knowledge regarding the roles of miRNAs in the ONH. The purpose of this study was to determine: (a) differences in the expression of profibrotic and anti-fibrotic miRNAs in normal ONH astrocytes treated with or without TGFβ2 and (b) whether candidate miRNAs (miR-200b-3p and miR-211-5p) regulate the expression of ECM and ECM associated proteins in the ONH.

METHODS: Primary normal human ONH astrocytes (ONA) were grown to 100% confluency. ONA were treated with 5ng/ml TGFβ2 or with control for 24hrs. miRNA qPCR arrays were performed to compare the expression levels of profibrotic and anti-fibrotic miRNAs in normal ONA treated with or without TGFβ2. ONA were transfected with miR-200b-3p and miR-211-5p mimics at 10nM, 5nM, and 1nM concentrations to confirm target predictions based on the TargetScan database. An all stars negative control siRNA was included which will not recognise any mammalian gene.

RESULTS: The miRNA qPCR arrays analysed from normal ONA exposed to TGFβ2 showed that TGFβ2 downregulated the expression of hsa-miR-200b-3p and hsa-miR-211-5p. Transfection of miR-200b-3p mim downregulated fibronectin (FN), gremlin, and tissue transglutaminase II in ONA. Transfection of miR-211-5p downregulated FN and gremlin in ONA .

CONCLUSIONS: Our results suggest that TGFβ2, which is elevated in the glaucomatous ONH, modulates the expression of miRNAs in ONA. These miRNAs target FN, TGM2, and gremlin to modify the ECM in the ONH. Downregulation of anti-fibrotic miRNAs may contribute to fibrosis of the ONH.