【作者】 李晓宁；

【导师】 肖君华； 周宇荀；

【作者基本信息】 东华大学 ， 化学， 2021， 博士

【摘要】 性发育是哺乳动物性成熟并获得生殖能力的过程,受神经-内分泌系统的精密控制。Kiss1/Kiss1R组成性发育的门控系统,它们将机体内外的调控信号整合后输入GnRH神经元。从下丘脑分泌到垂体的GnRH是神经系统输出的调控个体性发育和生殖能力的终极信号。下丘脑-垂体-性腺轴的再次活化以及Gn RH脉冲式分泌是性发育启动的标志。近些年,随着物质生活水平的提升,性早熟已经成为发病率最高的儿童内分泌疾病。解析调控性发育的遗传机制,对于预防性早熟和治疗生殖相关疾病的意义重大。本研究关注由基因转录后调控因子micro RNAs参与Kiss1/Gnrh1基因介导的性发育调控机制。分别以这两个性发育关键基因为中心,本课题主要开展了以下两部分的研究工作。第一部分的主题是研究参与Kiss1基因表达调控的miRNAs。为了便于高效地从全基因组范围筛选参与Kiss1基因表达调控的miRNAs,我们构建了一个Kiss1基因表达双色荧光报告系统。我们使用CRISPS/Cas9基因编辑技术将绿色荧光蛋白的编码基因e GFP插入到Kiss1基因终止密码子上游,使之享用全套Kiss1基因表达调控元件,以绿色荧光强度表征Kiss1表达水平。为了增强系统的特异性和稳定性,我们还为系统引入了提供内参荧光信号的红色荧光蛋白编码基因td Tomato的表达元件。借助流式细胞术,我们建立了灵敏高效的Kiss1基因表达调控因子检测方法。为提高功能miRNAs的筛选效率,我们先用生物信息学算法对小鼠全基因组miRNA文库进行简化。结合下丘脑miRNAs表达数据以及Target Scan和miRBase数据库中miRNAs的注释信息,综合考虑miRNAs的表达水平、调控靶基因的能力以及进化保守性,逐步缩小用于实验筛选的miRNAs范围。经过双色荧光报告系统和分子生物学实验筛选后,我们发现了在体外抑制Kiss1基因表达,并且在性发育期间下丘脑中表达水平持续降低的miR-199-3p。因此,将miR-199-3p作为候选Kiss1表达调控因子对分子机制进行进一步研究。在进行miR-199-3p调控Kiss1表达和性发育的机制研究时,我们将介导调控作用的信号通路作为研究对象。我们发现miR-199-3p通过协同抑制4个位于p38 MAPK通路中的蛋白激酶,高效调控信号通路活性。p38 MAPK通路活性的降低直接导致Kiss1转录因子活性和Kiss1基因表达水平的下降。无论是抑制大鼠下丘脑p38 MAPK通路活性,还是在小鼠下丘脑弓形核区特异性过表达miR-199-3p,都会引起个体性发育延迟、血清促性腺激素水平下降以及性腺发育受阻。第二部分的主题是解析miR-29对Gnrh1基因表达的调控作用。分析性发育期间下丘脑miRNAs表达谱,我们发现了在不同性发育阶段差异表达的miR-29。有研究显示,miR-29与个体生殖能力有关。因此,我们将miR-29作为参与性发育调控的候选基因进行功能解析。miR-29家族有四个成员,家族成员之间潜在的功能冗余可能为其功能解析带来挑战。为解决这一难题,我们构建了基于CRISPR/Cas9基因编辑技术的多基因共敲除单质粒系统,并将其应用于构建敲除任意组合miR-29家族成员的细胞模型。我们发现在miR-29家族成员全部缺失的GT1-7细胞中,性发育重要基因Gnrh1的表达水平上升2-3倍。经过生物信息学算法预测和分子生物学实验验证,我们发现miR-29-3p的靶基因Tbx21介导了miR-29家族对Gnrh1的转录调控。作为Gnrh1的转录激活因子,TBX21既可以直接结合于Gnrh1基因启动子直接发挥调控作用,又可以通过促进Gnrh1另一个转录因子Dlx1的表达间接发挥作用。我们还观测了干扰性发育前雌性小鼠下丘脑miR-29-3p和Tbx21的表达水平对个体性发育的影响。研究发现,下丘脑定位注射miR-29-3p抑制剂导致小鼠性发育提前。下丘脑视前区特异性Tbx21过表达小鼠不仅在青春期的性发育进程提前,而且在成年期个体的动情周期、血清促性腺激素水平和生殖能力也受到不同程度的影响。综上所述,我们的研究解析了两种新型的miRNAs介导的性发育调控机制,进一步拓展了调控性发育的遗传网络。根据miRNAs调控靶基因的特性,我们还建立了两种功能研究策略:其一,针对miRNAs的多靶点性和微效性,我们将由多组分构成、高度动态化且具有剂量敏感性的信号通路作为研究对象,证实了miRNAs可以通过多靶点协同调控同一生命过程;其二,针对miRNA家族成员之间的功能冗余,我们建立了多基因共敲除方法,实现在家族成员全部缺失的条件下进行基因功能研究。这两种策略克服了miRNAs功能研究的困难,可为其他miRNAs功能研究提供参考。更多还原

【Abstract】 Puberty is a key transitional period when sexual maturity and reproductive capacity are achieved.A sophisticated neuroendocrine network is involved in controlling this process.Acting as a gatekeeper,the Kiss1/Kiss1 R system integrates the regulatory signals inside and outside the body then input to GnRH neurons.GnRH secreted from the median eminence of the hypothalamus to the pituitary represents the final output of neuroendocrine system that regulates sexual development and reproductive function and its pulsatile secretion is the hallmark of puberty onset.For the improved living conditions,precocious puberty has become the most common endocrine disease in children.Clarifying the genetic mechanisms regulating the puberty onset is of great significance for preventing precocious puberty and treating reproductive-related diseases.Our study focused on Kiss1 or Gnrh1 gene involved mechanisms that were mediated by micro RNAs,a kind of posttranscriptional regulatory factors.Centering on these two key genes respectively,our works consist of the following two independent parts.The topic of the first part is the miRNAs involved in the regulation of Kiss1 expression.In order to screen the miRNAs involved in the regulation of Kiss1 expression from the whole genome efficiently,we constructed a dual fluorescent reporter system for Kiss1.In this system,the coding sequence of e GFP was inserted into Kiss1 locus,sharing the all regulatory elements of Kiss1.The level of Kiss1 expression was characterized by green fluorescence intensity.For the specificity and stability of this system,the td Tomato cassette was also introduced into this system who provided the reference fluorescence.With the help of flow cytometry,we established a highly sensitive and rapid detection program for the regulators of Kiss1 gene expression.In order to improve the screening efficiency of functional miRNAs,we simplified the mouse genome-wide miRNA library by bioinformatics algorithms first.Considering their expression levels,target numbers and evolutionary conservation,the scope of miRNAs for experimental screening was gradually narrow according to a combined analysis of miRNAs expression profile of hypothalamus and the annotation of the miRNAs in the Target Scan and miRBase database.After screened by the dual fluorescence reporter system and molecular biology experiments,only miR-199-3p which showed a steady trend of downregulation in the hypothalamus of mice from the infant stage to the adult stage inhibited the expression of Kiss1 in vitro.Therefore,the molecular mechanism of miR-199-3p,as a candidate Kiss1 expression regulator,was explored.In the study of the regulation mechanism,the signal pathway which mediated the effect of miR-199-3p on Kiss1 expression and puberty onset was analyzed.miR-199-3p were found to regulate the activity of p38 MAPK pathway by synergistically inhibiting four targets located in this pathways.While the reduced activity of p38 MAPK pathway lead to the decreased transcription of Kiss1.Either inhibiting the activity of the p38 MAPK pathway in the rat hypothalamus or overexpressing miR-199-3p in the mouse hypothalamus caused the delay of individual puberty onset,the decreased levels of serum gonadotropin and the postponed gonadal development.The subject of the second part is the effect of miR-29 on the expression of Gnrh1.By analyzing the expression profiles of hypothalamic miRNAs during sexual development,miR-29 was found to be differentially expressed at different stages of puberty onse in mouse hypothalamus.Moreover,several studies suggested that miR-29 is related to individual reproductive ability.Therefore,miR-29 was considered as a candidate gene for the regulation of puberty onset.The functional redundancy possiblely exist among four miR-29 family members brought challenges to our research.We established a scheme to knockout multiple genes simultaneously based on the CRISPR/Cas9 system to knockout all member of miR-29 family in one cell.And a series of cell models with any combination of miR-29 family member knockout were constructed using this scheme.With the absence of all miR-29 family member,the expression level of Gnrh1 increased 2-3times in GT1-7.Through the bioinformatics algorithm prediction and molecular biology experimental verification,Tbx21,the target gene of miR-29-3p,was found to mediate the effect of miR-29 family on the Gnrh1 expression.As one of the transcriptional activators of Gnrh1,TBX21 stimulates the expression of Gnrh1 directly by binding to its promoter region,and indirectly by activating the expression of Dlx1,another transcription activator of Gnrh1.Finally,we verified the effect of disturbing hypothalamic miR-29-3p and Tbx21 on the puberty onset of immature female mice.Stereotactic brain infusion of miR-29 inhibitor into the hypothalamus caused earlier puberty onset in prepubertal female mice than that of intact controls.The female mice with ectopic expression of Tbx21 in the hypothalamus were affected in not noly the puberty onset,but also the estrous cycle,serum gonadotropin level and fertility.In a word,our research explained the regulation mechanism of puberty onset involved in two novel miRNAs.Moreover,we provided two alternative strategies for functional miRNA analysis according to miRNA characters: one aimed at the multi-targeting and minor effect of miRNAs.Mediating by the signal pathways which were highly dynamic and exhibited a significant dosedependent response,miRNAs could coordinately regulate one biological process through multiple targets.And the other was a strategy against the functional redundancy among miRNA family members.A scheme to knockout multiple genes simultaneously was constructed.Therefore,the functional analysis of miRNA family could be carried out with the absence of all members.These two strategies overcomed the challenges of miRNAs function analysis and provided references for the research of other miRNAs function.更多还原