miR-31与多发性硬化

构建miR-31条件性基因敲除小鼠模型，在CD4+ T 细胞中条件性敲除 miR-31，可以促进多发性硬化的小鼠模型外周调节性T细胞的产生，进而减缓多发性硬化病的发生发展。进一步的研究表明，miR-31 通过靶向 G蛋白偶联受体家族中的Gprc5a的 3‘UTR 区域发挥其生物学作用。Gprc5a 缺失会影响诱导性调节性T细胞的形成，并且会加重多发性硬化病的发生发展，研究提出miR-31可以通过靶向Gprc5a负向调节外周调节性T细胞的分化。这一发现揭示了表观遗传调控调节性T细胞的新机制，可能为包括多发性硬化病在内的自身免疫性疾病的临床治疗提供新的靶标。

Fig1. Alleviation of autoimmune disease in cKO mice.(a) Schematic representation of the miR-31 locus and targeting strategy. Cre-mediated recombination of loxP sites in mice. Primers (P1 and P2) spanning the loxP sites were designed for genotyping floxed allele (1,195 bp) and deleted allele (474 bp). (b) PCR products of splenocytes and sorted CD4+ T cells derived from either miR-31fl/fl control or miR-31fl/flCD4Cre (cKO) mice. (c) qPCR analysis to confirm the deletion of miR-31 in CD4+ T cells derived from cKO mice. (d,e) Clinical scores and weight loss (mean±s.e.m.) of miR-31fl/fl or cKO mice after the induction of EAE were assessed every day (n=7 per group). ***P<0.001, two-tailed Student’s t-test for c, one-way analysis of variance for d and e. Data are representative of two (c) or three (b,d and e) independent experiments (mean±s.e.m.).

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miR-31与银屑病

miR-31也是银屑病治疗的潜在靶点。在银屑病病人和银屑病小鼠模型中，表皮中高度活化的NF- B信号分子促进角质形成细胞中miR-31的转录进而使之呈现高表达，高表达的miR-31直接靶向作用于细胞周期负向调控元件Ppp6c，导致角质形成细胞分裂加快进而过度增殖，从而促进了银屑病皮损区域表皮增生的病理过程。

构建miR-31条件性基因敲除小鼠模型，在小鼠表皮中条件性剔除miR-31，小鼠银屑病样皮损减轻，表皮增生状况缓解，真皮炎症细胞浸润数减少。

Fig2. Decreased epidermal hyperplasia and dermal cellular infiltrates in miR-31fl/fl/K5-Cre mice treated with IMQ. (a) Schematic representation of primers for genotyping and targeting strategy. (b) miR-31 genotyping using P1/P2, 1,064 bp band for miR-31fl/fl and 235 bp band for miR-31fl/fl/K5-Cre (cKO). DNA samples were prepared from total skin. (c) Cre-mediated tissue-specific deletion of miR-31 in epidermis. DNA samples were prepared from either epidermis or dermis. (d) miR-31 expression in epidermis derived from miR-31fl/fl and cKO mice. (e) Phenotypic presentation of mouse back skin for miR-31fl/fl or cKO mice treated with IMQ or vehicle for 7 days. (f) Splenomegaly and lymphadenopathy in miR-31fl/fl or cKO mice treated with IMQ or vehicle for 7 days. Data are representative of more than five mice. (g) Skin thickness was measured on the days indicated. Symbols represent mean skin thickness±s.e.m. for five to six mice per group. (h) H&E staining of the back skin of miR-31fl/fl or cKO mice treated with IMQ or vehicle. Dotted line indicates the border between the epidermis and the dermis. Scale bar, 100 μm. (i) Acanthosis of miR-31fl/fl or cKO mice treated with IMQ or vehicle. (j) Dermal cellular infiltrates of miR-31fl/fl or cKO mice treated with IMQ. (k) Immunostaining of Ki67 in lesional skin derived from miR-31fl/fl (left panel) or cKO (right panel) mice treated with IMQ. Scale bar, 100 μm. (l) Quantitation of Ki67+ cells in epidermis. For all measurements, numbers of specifically stained Ki67+ cells in epidermis counted in three high-power fields for each section were used. (m) qPCR analysis of epidermal differentiation markers (Keratin 10, Loricrin and Filaggrin) in miR-31fl/fl or cKO mice treated with IMQ or vehicle. Results (d,m) are presented as the ratio of miRNA to the small nuclear RNA U6 or of mRNA to the β-actin, relative to that in miR-31fl/fl mice. *P<0.05, **P<0.01, ***P<0.001, two-tailed Student’s t-test for d,i,j,l,m, one-way ANOVA for g. Data (g,i,j,l,m) are representative of two independent experiments with four to six mice per group in each (mean and s.e.m.).

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