miR-22转录后调控毛囊退化

发布时间：2018-06-24 23:17

本文选题：microRNA + miR-22　；参考：《中国农业大学》2015年博士论文

【摘要】：毛囊是皮肤的衍生物,它的生长发育具有周期性,即不断地经历由生长期向退行期和静止期转变,再由静止期转向下一个周期的生长期的循环过程。毛囊从生长期向退行期和静止期的转变,实际上是一个毛囊退化的生理过程,这个过程受到包括凋亡和生长抑制信号等通路的调控。但是,生长期末期毛囊细胞分化与毛干装配形成后毛囊退化过程的具体分子机制还不十分清楚。microRNA(miRNA)是一类20-22个核苷酸大小的非编码RNA,可以与靶基因的3’UTR结合而起到抑制靶基因的功能。近年来,越来越多研究报道miRNA在毛囊的形态发生,毛囊干细胞的增殖、分化和凋亡的稳态平衡等过程中都发挥着重要作用。已有研究报道表明进化上高度保守的miR-22在退行期和静止期的表达显著高于生长期。然而,miR-22是否在毛发周期和毛囊退化过程中发挥重要功能需要进一步探讨。本研究首先利用qPCR和原位杂交技术验证了miR-22在退行期和静止期高表达的表达模式。为了进一步揭示miR-22在调控毛囊发育和毛发周期方面的功能,本研究构建了可诱导的皮肤特异性过表达miR-22的转基因小鼠K14-rtTA/TRE-miR-22,购买了miR-22敲除鼠。通过体内实验显示,miR-22过表达可以促进毛囊从生长期向退行期转变,延迟毛囊从静止期向下一个周期的生长期的转变,从而导致小鼠脱毛。与过表达结果相反,miR-22敲除导致小鼠毛囊延迟从生长期进入退行期,加快从静止期向下一个生长期的转变。采用短期诱导miR-22表达的方法证实miR-22可以直接促进毛囊退化过程。进而,揭示miR-22可以通过抑制细胞增殖、细胞迁移、细胞分化和干细胞自我更新,同时促进细胞凋亡的多种途径来促进毛囊退化过程。在分子机制方面,本研究通过GO分析发现miR-22过表达小鼠毛囊生长期(P28,Dox P21)的基因表达谱与野生型小鼠毛囊退行期和静止期基因表达特征非常相似,即大量毛囊分化相关的转录因子基因、角质化蛋白基因、非膜结构细胞器基因、凋亡负调控因子基因以及细胞周期相关基因都显著下调。这说明miR-22是通过下调这些基因,特别是下调毛囊分化相关的转录因子基因和凋亡负调控因子基因的表达水平,促进了毛囊退化。为了揭示miR-22如何下调这些基因,本研究利用生物信息学方法分析发现：下调的毛囊分化相关的转录因子和凋亡负调控因子3'UTR中含有miR-22结合位点,是miR-22的潜在靶基因。通过双荧光素酶报告基因检测实验进一步验证了毛囊分化相关的转录因子Dlx3、Foxn1和Hoxcl3,以及BMP信号通路抑制因子Sostdc1是miR-22的功能靶基因。这些转录因子可以直接调控角蛋白基因的表达来影响内根鞘和毛干的形成。综上所述,本研究揭示miR-22是毛囊退化的重要转录后调控因子,可能成为临床上治疗脱发相关疾病的靶标分子。
[Abstract]:Hair follicles are derivatives of the skin, and their growth and development are cyclical, that is, they are constantly changing from growth phase to receding phase and stationary phase, and then from stationary stage to next cycle. The transition of hair follicles from growth stage to receding stage and stationary stage is actually a physiological process of hair follicle degeneration which is regulated by pathways including apoptosis and growth inhibition signal. However, The molecular mechanism of hair follicle degeneration after the differentiation of hair follicle cells and the formation of hair stem assembly at the end of growth period is not well understood. MicroRNA (miRNA) is a class of non-coding RNAs of 20-22 nucleotides, which can bind to the 3UTR of the target gene and play an inhibitory role. The function of target gene. In recent years, more and more studies have reported that miRNA plays an important role in the process of hair follicle morphogenesis, hair follicle stem cell proliferation, differentiation and homeostasis of apoptosis. It has been reported that the expression of miR-22, which is highly conserved in evolution, is significantly higher in receding and stationary phases than in growth period. However, whether miR-22 plays an important role in hair cycle and hair follicle degeneration needs further study. In this study, qPCR and in situ hybridization techniques were used to verify the high expression patterns of miR-22 in receding and stationary phases. In order to further reveal the function of miR-22 in regulating hair follicle development and hair cycle, a transgenic mouse, K14-rtTA-TRE-miR-22, which can induce skin specific expression of miR-22, was constructed, and miR-22 knockout mice were purchased. In vivo experiments showed that the overexpression of miR-22 could promote the transition of hair follicles from growth stage to receding stage, and delay the transition of hair follicle from stationary stage to next cycle, thus leading to hair loss in mice. Contrary to the overexpression results, the knockout of miR-22 resulted in delayed hair follicle transition from growth stage to retrogression phase, and accelerated the transition from stationary stage to next growth phase. Short-term induction of miR-22 expression showed that miR-22 could directly promote the process of hair follicle degeneration. Furthermore, it is revealed that miR-22 can promote hair follicle degeneration by inhibiting cell proliferation, cell migration, cell differentiation and self-renewal of stem cells, as well as promoting apoptosis. In terms of molecular mechanism, the gene expression profiles of miR-22 overexpression mouse hair follicle growth phase (P28 Dox P21) were similar to those of wild-type mouse hair follicle in receding and stationary stages by go analysis. Many transcription factor genes related to hair follicle differentiation, keratinizing protein genes, non-membrane organelle genes, apoptosis negative regulatory factor genes and cell cycle related genes were significantly down-regulated. This suggests that miR-22 promotes hair follicle degeneration by down-regulating the expression of these genes, especially the transcription factor related to hair follicle differentiation and the expression of apoptosis negative regulator gene. In order to reveal how miR-22 down-regulates these genes, bioinformatics analysis showed that down-regulated transcription factors related to hair follicle differentiation and down-regulated negative regulation factor 3UTR contain miR-22 binding sites, which are potential target genes of miR-22. The detection of double luciferase reporter gene further confirmed that Dlx3 Foxn1 and Hoxcl3, and that BMP signaling pathway inhibitor Sostdc1 is the functional target gene of miR-22. These transcription factors can directly regulate the expression of keratin gene to affect the formation of inner root sheath and hair stem. To sum up, miR-22 is an important posttranscriptional regulator of hair follicle degeneration and may be a target molecule for the treatment of alopecia related diseases.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R758.7

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