Huda, Syed Nazmul
(2022)
Microrna expression profiling in tears of children with vernal keratoconjunctivitis.
Masters thesis, Universiti Sains Malaysia.
Abstract
Vernal keratoconjunctivitis (VKC) is a chronic conjunctival inflammatory
condition usually affecting children. It is often present with severe manifestations
leading to corneal scar and blindness. Tears contain diverse concentrations of
microRNAs (miRNAs), which are small non-coding RNA molecules regulating various
cellular processes in various eye diseases. In this study we aim to generate miRNA
expression profile in tears of children with VKC in comparison to controls and evaluate
these miRNAs as the potential diagnostic biomarkers of VKC (phase II). However,
through a pilot study, we initially investigated the optimal miRNA quantity in tears
among three sampling groups, namely, unfractionated whole tears, whole-tear-derived
exosomes, and exosome-depleted (phase I). This was an experimental case-control
study conducted at Ophthalmology Clinic, Hospital Universiti Sains Malaysia, from
January 2020 till December 2020. Phase I of the study involved normal subjects while
phase II involved children with clinical diagnosis of VKC and control. RNA isolation
was performed using the miRNeasy Micro Kit and quantification through Bioanalyzer
RNA 6000 nano kit and Small RNA kit. VKC and control samples were screened for
miRNAs expression using Agilent microarray technique. The unfractionated whole
tears sampling group yielded better and optimal miRNA concentrations. Microarray
results revealed a total of 51 miRNAs that were differentially expressed among children
with VKC and controls. Out of these miRNAs, 48 were up-regulated and three were
down-regulated. The hsa-miR-1229-5p, hsa-miR-6821-5p, and hsa-miR-6800-5p were the three top up-regulated miRNAs, while the miRNAs, hsa-miR-7975, hsa-miR-7977,
and hsa-miR-1260a were the three down-regulated miRNAs. All the 48 up-regulated
miRNAs can be used as the potential diagnostic miRNA biomarkers for VKC due to
their higher discriminatory area under the curve (AUC) values. The miRNA target
prediction analysis has determined multiple gene targets out of which 16 overlapping
target genes (ARHGEF5, CCL22, CD276, LGALS9, MIF, PGF, PTGDS, PTGER,
B3GAT1, SOCS3, ICOSLG, TGM2, MMP25, NGFR, FOXP3, and HRH1) were
known to play role in causing conjunctival inflammation. The oxidative
phosphorylation, glycolysis/gluconeogenesis, and amino sugar and nucleotide sugar
metabolism were the top three KEGG pathways involved. In conclusion, miRNAs from
unfractionated whole tears were differentially expressed among children with VKC and
controls. Once validated, these miRNAs could serve as potential diagnostic biomarkers
for VKC and provides insights into the pathogenesis of VKC.
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