Expression of miR-486-3p in psoriatic lesions and its effect on keratin 17 expression
10.3760/cma.j.issn.0412-4030.2013.03.004
- VernacularTitle:MiR-486-3p在银屑病皮损的表达及对角蛋白17表达的影响
- Author:
Zhongbin SUN
;
Liang JIN
;
Erle DANG
;
Sen GUO
;
Chunying LI
;
Gang WANG
- Publication Type:Journal Article
- Keywords:
Psoriasis;
MicroRNAs;
Keratin-17;
MiR-486-3p
- From:
Chinese Journal of Dermatology
2013;(3):160-163
- CountryChina
- Language:Chinese
-
Abstract:
Objective To determine the expression of miR-486-3p in psoriatic lesions and healthy human skin and to estimate its effect on keratin 17 (K17) expression in HaCaT human keratinocytes.Methods Bioinformatics was used to predict microRNAs that may affect the expression of K17.Tissue samples were obtained from the lesions of 10 patients with psoriasis and normal skin of 10 healthy human controls.RNA was extracted from these tissue samples and reversely transcribed into cDNA with the addition of a Poly (A) tail.Then,real time quantitative PCR was performed to measure the expression of miR-486-3p.Cultured keratinocytes were transfected with miR-486-3p mimics or negative control,and Western blot was performed to determine K17 expression at 48 hours after the transfection.To evaluate the inhibitory effect of miR-486-3p on K17 expression,cultured 293T cells were transfected with the plasmid containing K17 3' untranslated region (UTR) seed sequence,three plasmids containing the complete deletion,interval mutation or double repeats of the seed sequence,or negative control plasmid.At 24 hours after the transfection,a dualluciferase reporter (DLR) assay was performed to quantify the expression of K17.Results Real time PCR showed that the expression level of miR-486-3p was significantly lower in psoriatic lesions than in the normal skin (0.211 ± 0.120vs.0.555 ± 0.425,t =2.62,v =9,P ＜ 0.05).The HaCaT cells transfected with the mimics of miR-486-3p exhibited decreased expression of K17 compared with those transfected with the negative control.DLR assay revealed that the expression level (fluorescence intensity) of K17 in the negative control group was significantly higher than that in the 293T cells transfected with the seed sequence and those with the double repeats of the seed sequence (100.00％ vs.65.31％ ± 6.32％ and 54.18％ ± 10.01％ respectively,both P ＜ 0.05),but did not differ from that in the 293T cells transfected with the complete deletion and interval mutation of the seed sequence (100.00％ vs.114.77％ ± 16.14％ and 110.21％ ± 12.99％ respectively,both P ＞ 0.05).Conclusions The expression of miR-486-3p,which may inhibit K17 expression by binding to the seed sequence of K17 3'UTR,is lower in psoriatic lesions than in normal skin.The decreased expression and inhibitory effect of miR-486-3p may be implicated in the initiation and progression of psoriasis.
