图1:mir - 675目标DUX4 ORF和3′UTR。一个RenLuc-DUX4-FL dual-luciferase记者和巨细胞病毒。段H19构造,U6。MIR675 H1。MIR675, U6。MIR675-3p, U6。MIR675-5p microrna。RenLuc-DUX4-FL包含DUX4 ORF和3′UTR序列融合Renilla终止密码子后的荧光素酶。外显子1 - 3(例1、2、3)表示。* SD5表示沉默突变Renilla神秘拼接捐赠者的荧光素酶。萤火虫荧光素酶作为转染控制。b U6。MIR675减少相对Renilla荧光素酶活动构造包含全身DUX4, DUX4 ORF,或者只DUX4 3 ' UTR(35±3%, 34±2%, 24±5%,分别;P < 0.0001)。 H19 reduced relative Renilla luciferase activity from the RenLuc-DUX4-FL by 36 ± 2% (P < 0.0001). All readings were normalized to Firefly luciferase and then to the milacZ negative control. U6.MIR675 did not reduce relative Renilla luciferase activity from the non-targeting RenLuc control plasmid (RenLuc). The U6.miDUX4.405 positive control is a published artificial microRNA with robust DUX4 silencing activity. Data indicate mean luciferase activity ± SEM (N = 3 independent experiments). c Relative Renilla luciferase activity was reduced in a dose-dependent manner by two miR-675 constructs: U6.MIR675 and H1.MIR675. Molar ratios of miRNA to reporter plasmid are indicated and data normalized to the miLacZ negative control. U6.MIR675 significantly reduced relative Renilla luciferase activity by 23 ± 3% (10:1 ratio), 29 ± 3% (20:1), and 34 ± 3% (40:1). H1.MIR675 performed better at lower doses, reducing relative Renilla luciferase activity by 42 ± 4%, 39 ± 5%, and 55 ± 3%. All samples were significantly different from the miLacZ control (P < 0.0001). Data represent mean normalized Renilla luciferase activity ± SEM (N = 4 independent experiments). d Droplet digital PCR (ddPCR). H1.MIR675 significantly reduced DUX4 mRNA levels by 71 ± 2% (P < 0.0001, ANOVA, N = 3) compared to the miLacZ negative control, 48 h following transfection of HEK293 cells with a 1:3 ratio of CMV.DUX4-FL/CMV.eGFP and H1.MIR675. Results represent the average absolute DUX4 mRNA concentration (copies/µL) ± 95% poisson confidence interval. Data represent mean relative concentration (copies/μL) ± SEM (N = 3 independent experiments). For b–d, One-way ANOVA followed by Dunnett’s multiple comparison tests were performed for statistical analyses. e Northern blot using RNA extracted from HEK293 cells transfected with the indicated expression plasmids. U6.miGFP is a negative control. Credit: DOI: 10.1038/s41467-021-27430-1
Facioscapulohumeral肌肉萎缩症(FSHD)是由在骨骼肌DUX4基因的异常表达。全国儿童医院的研究人员最近发布了一个内生的人类微rna, mir - 675,抑制DUX4表达和保护从DUX4-mediated肌肉细胞死亡在一个小鼠模型通过基因治疗管理。他们还表明,基于小分子治疗移植mir - 675抑制DUX4 mRNA和DUX4-associated生物标记肌管来自FSHD患者。
