CAJ | 학술논문

目的探讨银屑病患者皮损与健康人皮肤中miR-486-3p表达情况及其对角蛋白17(K17)表达的调控作用.方法采用生物信息学方法预测出调节K17表达的微RNA(microRNA),选取10例银屑病患者皮损和10例健康人皮肤提取RNA,用加PolyA尾法逆转录为cDNA,实时荧光定量PCR(qRT-PCR)进行荧光定量.用miR-486-3p模拟物(mimics)和阴性对照物(NC)转染人永生化角质形成细胞(HaCaT细胞),Western印迹检测K17表达情况；构建双荧光素酶报告系统,其中包括含有K173'UTR的片段,突变1组为将种子序列删除的片段,突变2组为将种子序列间隔突变的片段,突变3组为将种子序列重复2次的片段,并检测miR-486-3p是否与K17 3'UTR种子序列结合而抑制K17表达.结果银屑病皮损中miR-486-3P表达水平为0.211±0.120,低于健康对照的0.555±0.425,银屑病皮损为健康对照的0.380,两组比较,t=2.62,v=9,P＜0.05.Western印迹结果显示,miR-486-3p类似物转染HaCaT细胞后,K17表达水平明显降低.双荧光素酶报告系统检测结果,含有K173'UTR种子序列组与突变3组荧光强度分别为65.31±6.32和54.18±10.01,均低于对照组(P＜0.05)；突变1组和突变2组荧光强度分别为114.77±16.14和110.21±12.99,与对照组差异无统计学意义(P＞0.05).结论 miR-486-3p在银屑病皮损中的表达水平低于健康人皮肤,miR-486-3p通过与K17 3'UTR种子序列结合抑制K17表达,提示其表达水平降低及对K17调控作用的改变可能与银屑病的发生与发展相关.
목적 탐토은설병환자피손여건강인피부중miR-486-3p표체정황급기대각단백17(K17)표체적조공작용.방법 채용생물신식학방법예측출조절K17표체적미RNA(microRNA),선취10례은설병환자피손화10례건강인피부제취RNA,용가PolyA미법역전록위cDNA,실시형광정량PCR(qRT-PCR)진행형광정량.용miR-486-3p모의물(mimics)화음성대조물(NC)전염인영생화각질형성세포(HaCaT세포),Western인적검측K17표체정황；구건쌍형광소매보고계통,기중포괄함유K173'UTR적편단,돌변1조위장충자서렬산제적편단,돌변2조위장충자서렬간격돌변적편단,돌변3조위장충자서렬중복2차적편단,병검측miR-486-3p시부여K17 3'UTR충자서렬결합이억제K17표체.결과 은설병피손중miR-486-3P표체수평위0.211±0.120,저우건강대조적0.555±0.425,은설병피손위건강대조적0.380,량조비교,t=2.62,v=9,P＜0.05.Western인적결과현시,miR-486-3p유사물전염HaCaT세포후,K17표체수평명현강저.쌍형광소매보고계통검측결과,함유K173'UTR충자서렬조여돌변3조형광강도분별위65.31±6.32화54.18±10.01,균저우대조조(P＜0.05)；돌변1조화돌변2조형광강도분별위114.77±16.14화110.21±12.99,여대조조차이무통계학의의(P＞0.05).결론 miR-486-3p재은설병피손중적표체수평저우건강인피부,miR-486-3p통과여K17 3'UTR충자서렬결합억제K17표체,제시기표체수평강저급대K17조공작용적개변가능여은설병적발생여발전상관.
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.