Abstract
Purpose : Transforming growth factor beta 2 (TGFβ2) plays an important role in the pathogenesis of primary open angle glaucoma (POAG). TGFβ2 induces pathological changes in the trabecular meshwork (TM) and dysregulates microRNA (miRNA) expression. MiRNAs are cell and context specific epigenetic regulators of cellular functions and signalling pathways. Small RNA-sequencing was performed using cultured normal human TM cells treated with TGFβ2 to identify the TGFβ2 miRNAome.
Methods : Primary normal human TM cell cultures (n=5) were treated with TGFβ2 (5ng/mL) for 24 hours. RNA was extracted using Qiagen AllPrep kit and small RNA-sequencing was performed at the Genomics Core Facility in Queen’s University Belfast following QIAseq miRNA library prep with sequencing on the Illumina platform. Bioconductor was used for primary miRNA mapping and secondary differential expression. MiRNAs were analysed using miRTargetLink, TargetScan and miRTarBase to detect strong experimentally validated gene targets, and miRNAs with strong targets were run through KEGG analysis using DIANA mirPath v.3 software and miRPathDB to identify enriched pathways. MiRNA qPCR was performed to validate and replicate the small RNA-sequencing data in four independent biological samples for 10 miRNAs of interest.
Results : Significant (p<0.01) differential miRNA expression was seen for twenty-two miRNAs including miR-503, miR-145 and miR-143. At a significance level of p<0.05, seventy-two miRNAs were significantly altered of which five are implicated in the regulation of TGFβ signalling. qPCR confirmed significant differential miRNA expression in five miRNAs of interest. Enriched pathways regulated by these miRNAs included modulation of TGFβ signalling and the Hippo, MAPK and TNF signalling pathways. The identified SMAD-regulated miRNAs play roles in senescence, inflammation, and fibrosis.
Conclusions : Understanding the role of the TGFβ2 miRNAome in the TM will improve our understanding of physiological and pathological states in the TM. MiRNAs are epigenetic regulators of signalling pathways and the ability to therapeutically manipulate miRNAs with inhibitors or mimics may be an efficient therapeutic approach in POAG.
This is a 2021 ARVO Annual Meeting abstract.
Methods : Primary normal human TM cell cultures (n=5) were treated with TGFβ2 (5ng/mL) for 24 hours. RNA was extracted using Qiagen AllPrep kit and small RNA-sequencing was performed at the Genomics Core Facility in Queen’s University Belfast following QIAseq miRNA library prep with sequencing on the Illumina platform. Bioconductor was used for primary miRNA mapping and secondary differential expression. MiRNAs were analysed using miRTargetLink, TargetScan and miRTarBase to detect strong experimentally validated gene targets, and miRNAs with strong targets were run through KEGG analysis using DIANA mirPath v.3 software and miRPathDB to identify enriched pathways. MiRNA qPCR was performed to validate and replicate the small RNA-sequencing data in four independent biological samples for 10 miRNAs of interest.
Results : Significant (p<0.01) differential miRNA expression was seen for twenty-two miRNAs including miR-503, miR-145 and miR-143. At a significance level of p<0.05, seventy-two miRNAs were significantly altered of which five are implicated in the regulation of TGFβ signalling. qPCR confirmed significant differential miRNA expression in five miRNAs of interest. Enriched pathways regulated by these miRNAs included modulation of TGFβ signalling and the Hippo, MAPK and TNF signalling pathways. The identified SMAD-regulated miRNAs play roles in senescence, inflammation, and fibrosis.
Conclusions : Understanding the role of the TGFβ2 miRNAome in the TM will improve our understanding of physiological and pathological states in the TM. MiRNAs are epigenetic regulators of signalling pathways and the ability to therapeutically manipulate miRNAs with inhibitors or mimics may be an efficient therapeutic approach in POAG.
This is a 2021 ARVO Annual Meeting abstract.
| Original language | English |
|---|---|
| Article number | 1654 |
| Journal | INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE |
| Volume | 62 |
| Issue number | 8 |
| Publication status | Published (in print/issue) - Jun 2021 |
