miR-145靶向Crkl调控颗粒细胞激素合成的机制研究

Gynecologic endocrine diseases , such as PCOS, POI, abnormal uterine bleeding and so on, seriously affect the physical and mental health of patients and the quality of their life. Because of the complicated mechanism, the efficacy of treatment for these endocrine disorders remains limited. So We must move toward to explore underlying mechanisms, find new marker and therapy targets for these diseases. In our previous study, expression patterns of miRNAs in the neonatal mouse ovary were profiled by microarray, and 24 miRNAs whose abundances differed significantly between ovaries from 3- and 5-day-old mice were identified. Further study found that among these miRNAs, miR-145, which displayed the second highest degree among the up-regulated miRNAs, was an important miRNA that regulated the initiation of primordial follicle development and maintaind the dormancy of the primordial follicle pool. As the expression of miR-145 increased through the progress of follicle development, we propose that it may play an important role in the granulosa cell. Down-regulation of miR-145 could significantly inhibit cell proliferation, differentiation and hormone synthesis. Bioinformatics analysis and in vitro experiments confirmed that its target gene was the adaption protein gene Crkl, but its downstream specific regulatory mechanism is not clear and we want to figure out if it is a new predictive marker for ovary function and an effective target for these endocrine diseases. To answer these questions, in this project,we firstly intend to explore the role of CRKL in granaulosa cell, then find out the key protein which combined with it through IP and mass spectrometry, further reseach will figure out its targeting signaling pathway. Finally, miR-145 knockout mice will be used to clarify if miR-145 play an important role in the ovary function. We believe that investigating the functions of miR-145 in granulosa cell of the ovary will lead to an improved understanding of ovarian physiology and pathology, and further will provide a potential new marker for ovary function and target for theray of the endocrine diseases.

妇科内分泌疾病复杂的发病机制是导致其治疗艰难的根本原因之一，因此探讨卵巢细胞内分泌异常机制，寻找新的反映内分泌异常的分子标记及治疗靶点是该领域亟待解决的关键问题。在前期研究中我们通过miRNA芯片发现并首次阐明miR-145可调控始基卵泡募集，随后研究发现miR-145在小鼠卵巢生长卵泡颗粒细胞中高表达，下调其表达可显著改变细胞形态、抑制细胞增殖、分化与激素合成，综合生物信息学分析及体外实验证实连接蛋白Crkl基因为其靶基因，但其下游具体调控机制不明确。因此本项目拟继续探讨CRKL对颗粒细胞的作用，并筛选出与CRKL结合的关键蛋白及其调控的信号通路从而明确miR-145通过作用于Crkl、募集相关关键蛋白、改变颗粒细胞形态调节激素合成的确切机制。进一步采用基因敲除小鼠阐明miR-145缺陷对卵巢内分泌及生殖功能影响，从而为卵巢内分泌功能异常提供新的分子标记及有效的治疗靶点。

MicroRNAs(miRNAs)作为转录后水平的重要调控分子，越来越多的证据表明，miRNAs 主要通过作用于卵巢体细胞来调节卵巢功能, 如颗粒细胞。研究表明miRNAs可影响激素合成的各个环节。本课题组在前期研究中，首次发现在miR-145可调节始基卵泡的募集及发育。随着卵泡的募集，始基卵泡中颗粒细胞增殖、分化，而颗粒细胞中miR-145表达上调，这提示miR-145可能在颗粒细胞增殖、分化过程中发挥作用。颗粒细胞的分化是卵泡发育成熟的关键，如分化异常则可出现卵泡无法成熟、排卵，直接影响卵巢两大功能：内分泌与生殖功能。因此本课题拟探讨miR-145调控颗粒细胞激素合成的机制探。研究内容：包括1） miR-145调控颗粒细胞激素合成的机制探讨；2）验证该分子对卵巢生殖与内分泌功能的影响。重要结果：一、机制探讨：构建crkl载体，上调Crkl抑制颗粒细胞增殖，抑制颗粒细胞分化；下调CRKL表达可抑制孕激素合成，对雌激素合成无明显作用，同时影响激素合成相关关键酶的表达。下调miR-145与上调CRKL均可激活P38/JNK MAPK信号通路；二、鉴于该分子在始基卵泡募集及后续发育卵泡颗粒细胞中的关键作用进一步探讨其临床应用：探讨miR-145在POI患者和非POI对照人群血清中的表达，并分析其用作POI诊断的效能。与非POI不孕人群相比，POI患者血清中miR-145表达显著下调，差异具有统计学意义（p<0.01）。运用ROC曲线分析曲线下面积，miR-145的AUC为0.908 [95%CI = 0.838-0.977]。血清miR-145表达水平与血清FSH呈负相关（p<0.05），与T呈正相关（p<0.05），与AFC呈显著正相关（p<0.01）。上述研究结果提示：miR-145在颗粒细胞激素合成过程中扮演重要角色，而血清miR-145的表达水平可作为原发性卵巢功能低下早期预警和诊断的新的无创生物标志物。