PAK1调控人类精原干细胞自我更新与凋亡的作用及分子机制研究

Spermatogonial stem cells (SSCs) have significant applications in reproductive medicine and regenerative medicine. However, very little is known about genes in mediating the fate determinations of human SSCs, due to the limited source of human primary SSCs and difficulty in obtaining human testis tissues. In our preliminary studies, we revealed that PAK1 level was up-regulated by EGF but not by GDNF or FGF2. PAK1 knockdown led to the decrease in the proliferation and DNA synthesis of human SSC line in vitro and in vivo, whereas it enhanced cell apoptosis. RNA sequencing as well as cellular and molecular assays identified that PDK1, ZNF367, KDR, and miR-31-5p were targets of PAK1 in human SSC line. The levels of phos-ERK1/2 and phos-AKT were decreased by PAK1 siRNAs. Therefore, it is essential for us to complete the following important Aims: i) to uncover the function of PAK1 in regulating the self-renewal and apoptosis of human SSCs; ii）to seek the factors that mediate PAK1 in human SSCs as well as the signaling pathways and cell cycle proteins of PAK1 in regulating human SSCs; and iii) to identify the targets of PAK1 in controlling human SSCs and to explore the roles and interactions of these targets and PAK1 in human SSCs with the objective to draw the regulation network of PAK1 in controlling human SSCs. This grant is of particular significance, because it could provide novel gene regulation and network underlying the fate decisions of human spermatogonial stem cells, and it would offer important scientific basis for the application of human spermatogonial stem cells in reproductive medicine and regenerative medicine.

精原干细胞在生殖医学和再生医学中有重要的应用前景。然而，基因对人类精原干细胞的作用及调控机理尚不清楚。我们前期研究发现，EGF增高人精原干细胞系PAK1表达水平；PAK1敲低减少人精原干细胞系增殖和DNA合成，增加其凋亡；PAK1调控人精原干细胞系的PDK1、KDR、ZNF367、miR-31-5p以及ERK1/2和AKT信号通路。因此，本项目拟开展如下研究内容：1）揭示PAK1调控人精原干细胞自我更新与凋亡的功能；2) 发现调控人精原干细胞PAK1的因子，确定PAK1调控人精原干细胞的信号通路和细胞周期蛋白; 3) 明确PAK1调控人精原干细胞的下游靶基因和miRNA，探讨PAK1与这些靶基因和miRNA相互作用及其功能，绘制PAK1调控人精原干细胞的网络图。本项目将为解析人类精原干细胞命运决定的机制提供新的遗传调控机理，对人类精原干细胞在生殖医学与再生医学的应用有重要意义。

精原干细胞在生殖医学和再生医学中有重要的应用前景。然而，基因对人精原干细胞的作用及调控机理尚不清楚。因此，本项目开展以下研究内容：1）PAK1调控PDK1/KDR/ZNF367和ERK1/2 及AKT信号通路促进人精原干细胞的增殖并抑制其凋亡。EGF可增加人精原干细胞中PAK1的表达水平。PAK1敲低后抑制人类精原干细胞DNA合成和增殖，促进其凋亡。人精原干细胞系异种移植实验显示，PAK1可以促进精原干细胞系在小鼠睾丸内的增殖，抑制其凋亡。RNA-seq表明，PAK1敲低后，与细胞增殖密切相关的PDK1、ZNF367和KDR的表达水平显著降低。免疫共沉淀(Co-IP)表明，PAK1可以与PDK1相互作用；ZNF367敲低后，PDK1和KDR mRNA表达水平发生降低。PDK1-、KDR -和ZNF367-siRNAs转染后，人精原干细胞增殖受到抑制，细胞凋亡发生率增加；PAK1敲低后，phos-ERK1/2、phos-AKT和cyclin A的蛋白水平降低。与梗阻性无精子症人群相比，PAK1的mRNA和蛋白表达水平在非梗阻性无精子症人群中表达降低。2）MiRNA-31-5p通过靶向JAZF1参与PAK1引起的人精原干细胞的增殖、DNA合成和凋亡。芯片检测及Real-time PCR论证，PAK1敲低后，人精原干细胞 miR-31-5p显著升高。PAK1敲低后，miR-31-5p表达显著升高。miR-31-5p上调抑制人精原干细胞增殖和DNA合成。miR-31-5p增强人精原干细胞凋亡。JAZF1是miR-31-5p的靶标，JAZF1沉默降低精原干细胞的增殖、增强其凋亡率；miR-31-5p高表达可减少细胞周期蛋白Cyclin A2。3）通过RNA测序与分子生物学实验发现，PAK1下游基因RNF144B、OIP5、YAP1调控人类精原干细胞中的命运机制。4）过表达DAZL家族三个基因诱导人类支持细胞重编程成为具有自我更新与分化潜能的精原干细胞。5）miR-1908-3和miR-122-5p 调控人精原干细胞自我更新与凋亡的功能及其靶基因。本项目将为解析人类精原干细胞命运决定的机制提供新的遗传调控机理，为治疗男性不育症提供新的靶点。