Kamal, Nik Nur Syazana Nik Mohamed
(2021)
Analysis of microrna derived from saliva- and plasma-exosome as potential biomarker(s) for chronic periodontitis.
PhD thesis, Universiti Sains Malaysia.
Abstract
Chronic periodontitis (CP) is an oral cavity disease arising from chronic
inflammation of the periodontal tissues. Exosomes are lipid vesicles enriched in
specific microRNAs (miRNAs), potentially providing a disease-specific diagnostic
signature. miRNAs have been demonstrated to play an important role in regulating
the immuno-inflammatory response; however, the function of salivary and plasma
exosomes miRNAs in chronic periodontal inflammation has not been investigated.
To assess the value of exosomal miRNAs as potential biomarkers for CP, miRNA
expression were profiled using Agilent miRNA microarray platform. From 2,549
tested miRNAs, 1,985 were significantly down-regulated and 10 significantly upregulated
in CP saliva samples; whereas 33 miRNAs were significantly downregulated
in CP plasma samples; as compared to respective healthy samples.
Expression of hsa-miR-5006-5p, hsa-miR-942-3p, hsa-miR-30e-3p and hsa-miR-
199a-3p was validated using reverse transcription quantitative polymerase chain
reaction (RT-qPCR) method. RT-qPCR results recorded significant replicable
miRNAs profiling results, strengthening the potential of these miRNAs to be
developed into biomarker(s) for the disease. For in vitro study, human gingival
fibroblasts (HGFs) cells were induced with lipopolysaccharides (LPS) from
Porphyromonas gingivalis to mimic inflammation condition, whereas the miRNA
mimics were used for transfection. hsa-miR-30e-3p mimic was transfected either
before or after induction of LPS to test the reaction of the mimic towardsinflammation condition. hsa-miR-30-3p level was only significantly up-regulated
after the induction, resembling the ability of the mimic to act as healer rather than as
a shield. Lastly, the hsa-miR-5006-5p and hsa-miR-30e-3p mimics were transfected
into HGFs cells and expression of bone morphogenetic protein receptor type II
(BMPR2), interleukin 1 receptor 1 (IL1R1), nuclear factor kappa B subunit 1
(NFκB1) and TLR4 were quantified. The BMPR2, IL1R1, NFκB1 and TLR4 were
found significantly down-regulated in both types of transfected cells (either
transfected with hsa-miR-5006-5p-mimic or hsa-miR-30e-3p-mimic). Yet, only the
significant down-regulation of BMPR2 in transfected cells-with hsa-miR-5006-5pmimic
parallel with extrinsic inflammation effect, making BMPR2 as the direct target
gene for hsa-miR-5006-5p. Meanwhile, the significant down-regulation of IL1R1,
NFκB1 and TLR4 in transfected cells-with hsa-miR-30e-3p-mimic parallel with
opposite extrinsic inflammation effects, making them as the potential direct target
genes for hsa-miR-30e-3p. Although extensive mechanism study is necessary in
future, nevertheless, the replicable results from miRNAs profiling, RT-qPCR, and in
vitro study (especially for transfection of hsa-miR-30e-3p/ LPS induction
experiments), are nough to ensure the high potential of these miRNAs as
biomarkers for CP.
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