Keflee, Rashidi Dzul
(2018)
The effect of microrna targeting IL17RA in regulating the expression of RANKL and OPG in stem cells from human exfoliated deciduous teeth.
Masters thesis, Universiti Sains Malaysia.
Abstract
IL17A is now becoming a key role in bone remodelling. The regulations of
osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-β ligand
(RANKL) by IL17A were briefly explained in human mesenchymal stem cell (hMSC).
Herein, the aim of the present study was to determine the potential microRNA
targeting IL17RA and its effects toward OPG and RANKL expression in stem cell from
human deciduous teeth (SHED). Potential microRNA was predicted by using three
latest algorithmically programs such as DIANA-micro T CDS, TargetScan v7.1 and
miRWalk v2.0 through a complex filtration process via in silico. The concentration of
microRNA-targeting GAPDH was optimised for the most efficient downregulation of
GAPDH mRNA. The concentrations of 25, 50 and 100 nM and transfection duration
of 48 hours were tested with microRNA-mimic negative control (microRNA-mimic
NC) as a negative control. The results showed the GAPDH mRNA level was lowest at
concentration of 50 nM after evaluated by quantitative real time PCR and normalised
with β-actin. This concentration can be used as the general miRNA concentration in
this study. SHED was cultured in complete alpha minimum essential media (αMEM)
supplemented with osteogenic media (OM) and treated with 50 ng/mL of recombinant
interleukin 17A (rIL17A) for 7 days (early osteogenic phase). Treated cells were then
transfected with 50 nM of positive control mimic, negative control mimic and
predicted microRNAs mimic from in silico study which are hsa-miR-6761-5p and hsamiR-
4524a-3p. Downregulation of target gene and its effects towards osteogenic
markers were evaluated by measuring the expressions of IL17RA, OPG, RANKL and
GAPDH by quantitative real-time PCR and normalised with β-actin. Hence,
transfections of both microRNA mimics showed downregulation of IL17RA gene
which hsa-miR4524a-3p showed stronger downregulation (p < 0.001) when compared
to hsa-miR-6761-5p (p <0.01). In addition, there was a significant downregulation of
OPG expression from both microRNAs and downregulation of RANKL was also noted
in both transfected SHED, however the reduction was not significant. These findings
demonstrate the role of microRNA targeting IL17RA in the expression of OPG and
RANKL in SHED, thus suggesting its important role in bone physiology of SHED.
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