Functions of Dicer, Drosha and miRNAs in the skin

描述(申请人提供)：microRNA在表皮和毛囊形态发生和动态平衡中的功能是皮肤生物学的一个令人兴奋的新领域，对我们理解皮肤和毛囊功能和皮肤疾病(包括脱发、皮肤肿瘤发生和伤口愈合缺陷)具有重要意义。本文提出的实验利用体内和体外技术来更精确地确定miRNAs在皮肤动态平衡中的全球作用，并开始识别对正常皮肤功能至关重要的特定miRNAs及其靶基因。因此，我们相信，我们的研究将对我们理解皮肤生物学具有重大意义，并有可能最终揭示从脱发到皮肤肿瘤等一系列疾病的治疗目标。最近发现的由microRNAs(MiRNAs)进行的转录后调控(MiRNAs)揭示了一种以前不为人知的机制，通过它来控制和精炼基因表达。非蛋白质编码miRNA前体(pri-miRNAs)由核基因转录而成，形成独特的茎环结构，可被核酶DROSHA及其辅因子Dgcr8特异性识别和切割，形成前miRNAs。前miRNAs从细胞核中输出，并被Diller酶加工成大约22个核苷酸的成熟miRNA。MiRNAs被组装成RISC复合体，RISC复合体通过抑制3‘非编码区含有部分互补序列的靶mRNAs的翻译和/或破坏其稳定性来调节基因的表达。已在哺乳动物中发现了数百个miRNAs，其中至少33个在皮肤中高度表达。小鼠独特的Disher基因的结构性缺失会导致成熟miRNAs的丧失和多种表型，包括：毛囊形态发生和维持失败；毛囊干细胞标志物缺乏表达；表皮-真皮信号中断；毛囊真皮乳头外翻；以及表皮过度增殖和凋亡。基于这些表型，我们假设DICER在出生后是维持毛囊生长和表皮动态平衡所必需的，它的部分功能是在毛囊干细胞中发挥作用。我们进一步假设，表皮Dice突变表型是由miRNA耗尽引起的。为了验证这些假说，我们将：(I)诱导性地删除出生后皮肤中的表皮DICER；(Ii)特异性地删除毛囊干细胞中的DICER；(Iii)确定另一个重要的miRNA加工途径组件的表皮枯竭是否复制了失去EFID的影响；(Iv)鉴定在不同皮肤间隔中表达的miRNAs，并开始确定它们的靶向mRNAs。

DESCRIPTION (provided by applicant): microRNA function in epidermal and hair follicle morphogenesis and homeostasis is an exciting new area of skin biology that has major implications for our understanding of skin and hair follicle function and skin disorders including alopecias, skin tumorigenesis, and wound healing defects. The experiments proposed here use in vivo and in vitro techniques to more precisely define the global roles of miRNAs in skin homeostasis, and to begin to identify specific miRNAs and their target genes that are critical for normal skin function. As such we believe that our studies will have major significance for our understanding of skin biology and have the potential to ultimately reveal therapeutic targets for a range of diseases from alopecias to skin tumors. The recent discovery of post-transcriptional regulation by microRNAs (miRNAs) revealed a previously unsuspected mechanism by which gene expression is controlled and refined. Non-protein coding miRNA precursors (pri-miRNAs) are transcribed from nuclear genes and form distinctive stem-loop structures that are specifically recognized and cleaved by the nuclear enzyme Drosha and its cofactor DGCR8 to form pre-miRNAs. Pre-miRNAs are exported from the nucleus and processed to approximately 22 nucleotide mature miRNA by the Dicer enzyme. miRNAs are assembled into the RISC complex which regulates gene expression by inhibiting the translation of, and/or destabilizing, target mRNAs that contain partially complementary sequences in their 3' non-coding regions. Several hundred miRNAs have been identified in mammals, and at least 33 are highly expressed in the skin. Constitutive epidermal deletion of the unique mouse Dicer gene results in loss of mature miRNAs and multiple phenotypes including: failure of morphogenesis and maintenance of hair follicles; absence of expression of hair follicle stem cell markers; disrupted epidermal-dermal signaling; evagination of hair follicle dermal papillae; and epidermal hyperproliferation and apoptosis. Based on these phenotypes we hypothesize that Dicer is required postnatally for maintaining hair follicle growth and epidermal homeostasis, and that it functions, in part, within hair follicle stem cells. We further hypothesize that epidermal Dicer mutant phenotypes result from miRNA depletion. To test these hypotheses, we will: (i) inducibly delete epidermal Dicer in postnatal skin; (ii) delete Dicer specifically within hair follicle stem cells; (iii) determine whether epidermal depletion of another essential miRNA processing pathway component replicates the effects of loss of epidermal Dicer; (iv) characterize miRNAs expressed in different skin compartments and begin to identify their target mRNAs.