Small RNA Sequencing Reveals a Distinct MicroRNA Signature between Glucocorticoid Responder and Glucocorticoid Non-Responder Primary Human Trabecular Meshwork Cells after Dexamethasone Treatment

Abstract

Glucocorticoids (GCs) are known to regulate several physiological processes and are the mainstay in the management of inflammatory eye diseases. The long-term use of GC causes raised intraocular pressure (IOP) or ocular hypertension (OHT) in about 30-50% of the susceptible individuals depending on the route of administration, and can lead to steroid-induced secondary glaucoma. The present study aims to understand the role of microRNAs (miRNAs) in differential glucocorticoid (GC) responsiveness in human trabecular meshwork (HTM) cells using small RNA sequencing. The human organ-cultured anterior segment (HOCAS) model was used to identify whether donor eyes were from GC-responders (GC-R; n = 4) or GC-non-responders (GC-NR; n = 4) following treatment with either 100 nM dexamethasone (DEX) or ethanol (ETH) for 7 days. The total RNA was extracted from cultured HTM cells with known GC responsiveness, and the differentially expressed miRNAs (DEMIRs) were compared among the following five groups: Group #1: ETH vs. DEX-treated GC-R; #2: ETH vs. DEX-treated GC-NR; #3: overlapping DEGs between Group #1 and #2; #4: Unique DEMIRs of GC-R; #5: Unique DEMIRs of GC-NR; and validated by RT-qPCR. There were 13 and 21 DEMIRs identified in Group #1 and Group #2, respectively. Seven miRNAs were common miRNAs dysregulated in both GC-R and GC-NR (Group #3). This analysis allowed the identification of DEMIRs that were unique to GC-R (6 miRNAs) and GC-NR (14 miRNAs) HTM cells, respectively. Ingenuity Pathway Analysis identified enriched pathways and biological processes associated with differential GC responsiveness in HTM cells. This is the first study to reveal a unique miRNA signature between GC-R and GC-NR HTM cells, which raises the possibility of developing new molecular targets for the management of steroid-OHT/glaucoma.

Keywords: glaucoma; glucocorticoids; intra-ocular pressure; miRNA; microRNAs; ocular hypertension; pathway analysis; small RNA-Seq; trabecular meshwork.

Figure 1

Volcano plot showing the distribution of DE miRNAs. The dys-regulated DEMirs of GC-responder and non-responder HTM cells are shown in (A,B) respectively. The fold of change (log2) and p-value (−log 10) of the dys-regulated miRNAs in DEX-treated cells compared to vehicle control are shown in volcano plot. Significant p-value < 0.05, only taken for consideration (red color).

Figure 2

Venn diagram showing differentially expression groupings. DE miRNAs of three groups from miRNA-seq data are shown. Only genes with absolute fold change 1.5 and significant p-value < 0.05 were included in these groupings. Group #1: DE miRNAs between ETH- and DEX-treated cells of GC-R HTM cells, Group #2: DE miRNAs between ETH- and DEX-treated cells of GC-NR HTM cells, Group #3: Overlapping DE miRNAs between Group #1 and Group #2; Group #4: uniquely expressed miRNAs in GC-R and Group #5: uniquely expressed DE miRNAs in GC-NR.

Figure 3

Validation of DE miRNAs by qPCR. Expression profile of selected miRNAs identified from miRNA-seq was validated by qPCR is shown [(i) hsa-miR 124-3p; (ii) hsa-miR 335-3p; (iii) hsa-miR 335-5p; (iv) hsa-miR483-3p; (v) has-miR 483-5p; (vi) hsa-miR 675-3p; (vii) hsa-miR 675-5p; (viii) hsa-miR 44853-3p and (ix) hsa-miR 549-a]. Primary HTM cells were treated with 100 nM DEX or 0.1% ETH for 7 days. Total RNA was extracted, converted to cDNA and the expression profile of selected miRNAs were carried out qPCR. miRNA expressions were normalized to RNU6 and analyzed using the 2^−ΔΔCT method [* p < 0.05].

Figure 4

Validation of miRNA sequencing by qPCR. Comparison of selected genes expression profile from Group #1 (i) and Group #2 (ii) miRNA sequence by qPCR; 2^ΔΔCt method was used for calculation of miRNA expression changes and U6 was used as an internal control.

Figure 5

Top 10 predicted KEGG pathways and GO biological processes of the ‘Target MRNA List 1’ of Group #3 (A), #4 (GC-R) (B), and #5 (GC-NR) (C). Dot colors: Log (1/p-value). Dot sizes: % genes in set. The combined score is defined based on the percentages of genes in set and Log (1/p-value) by [3]. p-value < 0.05 was considered to be statistically significant.

Figure 6

Interaction networks of the DEMIRs from Group #4 (GC-R) (A) and Group #5 (GC-NR) (B) (absolute LogFC > 2 and p < 0.05) and their negatively corelated target mRNAs (absolute FC > 2 and p < 0.05). Orange: up-regulated miRNAs/mRNAs. Blue: down-regulated miRNAs/mRNAs. Green circle: predicted pathways that the target mRNAs are located.

Figure 7

Heatmap shows the top 20 significant pathways (A) and biological processes (B) in the comparative analysis of Groups #3, #4 (GC-R), and #5 9GC-NR).