The effect of microrna targeting IL17RA in regulating the expression of RANKL and OPG in stem cells from human exfoliated deciduous teeth
Year: 2018
Type: dissertation
Abstract: IL17A is now becoming a key role in bone remodelling. The regulations of \nosteoprotegerin (OPG) and receptor activator of nuclear factor kappa-β ligand \n(RANKL) by IL17A were briefly explained in human mesenchymal stem cell (hMSC). \nHerein, the aim of the present study was to determine the potential microRNA \ntargeting IL17RA and its effects toward OPG and RANKL expression in stem cell from \nhuman deciduous teeth (SHED). Potential microRNA was predicted by using three \nlatest algorithmically programs such as DIANA-micro T CDS, TargetScan v7.1 and \nmiRWalk v2.0 through a complex filtration process via in silico. The concentration of \nmicroRNA-targeting GAPDH was optimised for the most efficient downregulation of \nGAPDH mRNA. The concentrations of 25, 50 and 100 nM and transfection duration \nof 48 hours were tested with microRNA-mimic negative control (microRNA-mimic \nNC) as a negative control. The results showed the GAPDH mRNA level was lowest at \nconcentration of 50 nM after evaluated by quantitative real time PCR and normalised \nwith β-actin. This concentration can be used as the general miRNA concentration in \nthis study. SHED was cultured in complete alpha minimum essential media (αMEM) \nsupplemented with osteogenic media (OM) and treated with 50 ng/mL of recombinant \ninterleukin 17A (rIL17A) for 7 days (early osteogenic phase). Treated cells were then \ntransfected with 50 nM of positive control mimic, negative control mimic and \npredicted microRNAs mimic from in silico study which are hsa-miR-6761-5p and hsamiR- \n4524a-3p. Downregulation of target gene and its effects towards osteogenic \nmarkers were evaluated by measuring the expressions of IL17RA, OPG, RANKL and \nGAPDH by quantitative real-time PCR and normalised with β-actin. Hence, \ntransfections of both microRNA mimics showed downregulation of IL17RA gene \nwhich hsa-miR4524a-3p showed stronger downregulation (p < 0.001) when compared \nto hsa-miR-6761-5p (p <0.01). In addition, there was a significant downregulation of \nOPG expression from both microRNAs and downregulation of RANKL was also noted \nin both transfected SHED, however the reduction was not significant. These findings \ndemonstrate the role of microRNA targeting IL17RA in the expression of OPG and \nRANKL in SHED, thus suggesting its important role in bone physiology of SHED.
Author: Rashidi Dzul Keflee
Language: English
Topic: Bone Metabolism and Diseases
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W3192447959