非编码小RNA调控正常与异常人精原干细胞自我更新与分化的作用及其机制研究

Spermatogenesis is a complex process by which spermatogonial stem cells self-renew and differentiate into mature sperm. Studies on spermatogonial stem cells are of great significance since they are the unique stem cells that transmit genetic information to subsequent generations. The fate determinations of spermatogonial stem cells are mainly regulated by endogenous molecules within the cells. However, it remains unknown about the roles and mechanisms of non-coding small RNA (miRNA) in regulating the self-renewal and differentiation of spermatogonial stem cells. We have explored the function and targets of miRNA-20 and miRNA-106a in mediating the proliferation and maintenance of mouse spermatogonial stem cells (Stem Cells, 2013, 31: 2205-2217). Furthermore, we have compared the global expression profiles of miRNAs among human spermatogonia, pachytene spermatocytes, and round spermatids, and we identified 599 miRNAs that are differentially expressed among these cells (Scientific Reports, 2015, 5: 8084). Notably, we have recently revealed, using RNA deep-sequencing and real time PCR, that 395 miRNA were differentially expressed in human spermatogonia between normal men and non-obstructive azoospermia (NOA) patients. Based upon these findings, it is essential and necessary for us to conduct this important project with the following Aims: i) to identify the differential expression and cellular localization of certain key miRNA, including has-miR-122-5p,has-miR-373-3p,has-miR-100-5p, and has-miR-145-5p, in human spermatogonial stem cells between normal men and NOA patients using Northern blots and the in situ hybridization; ii) to explore the roles of certain key miRNA, including has-miR-122-5p, has-miR-373-3p, has-miR-100-5p, and has-miR-145-5p, in regulating the self-renewal and differentiation of normal and aberrant human spermatogonial stem cells of NOA patients; and iii) to unveil the binding targets of these key miRNA and the function of their targets in mediating the fate determinations of normal and aberrant human spermatogonial stem cells of NOA patients. Significantly, this project could provide novel epigenetic regulatory mechanisms underlying human normal and abnormal spermatogenesis. This project is of particular significance, because it could offer novel endogenous targets for treating male infertility and preventing the birth defect of the offspring.

精子发生是指精原干细胞自我更新与分化为精子细胞的过程。精原干细胞的命运决定主要受细胞内源性分子的调控。然而，非编码小RNA（miRNA）对人类精原干细胞自我更新与分化的作用及其机理尚未见报道。申请人前期RNA深度测序显示，395种miRNA在正常与非梗阻性无精子症（NOA）病人精原细胞的表达差异显著。因此，本项目拟开展如下重要研究内容：1）发现和鉴定关键miRNA（如miR-122-5p、miR-373-3p、miR-100-5p、miR-145-5p）在正常与NOA病人精原干细胞的差异和定位表达；2）探讨关键miRNA对正常和NOA病人的精原干细胞自我更新与分化为精子细胞的作用；3）揭示关键miRNA对正常和NOA病人的精原干细胞自我更新与分化为精子细胞的调控靶标及其生物学功能。本项目将为阐释人类精子发生及其异常机制提供新的表观遗传调控机理，对治疗男性不育和预防子代出生缺陷有重要意义。

精原干细胞的自我更新、分化、凋亡主要受细胞内源性分子的调控。然而，非编码小RNA（miRNA）对人类精原干细胞命运决定的功能及其调控机理未见报道。在本项目中，我们完成了以下研究内容，取得重要研究结果：1）利用RNA深度测序，绘制非梗阻性无精子症（NOA）与梗阻性无精子症(OA)的精原细胞、粗线期精母细胞、精子细胞的miRNA大规模差异表达谱，预测和验证一些差异表达miRNA的靶标。2）MiRNA-663a靶向调控NFIX与作用于细胞周期蛋白A2、B1和E1促进人类精原干细胞自我更新、DNA合成并抑制细胞凋亡。3）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。4）miR-1908-3p通过KIF2调控人精原干细胞的增殖与凋亡。5）miR-122-5p靶向调控CBL并与长链非编码RNA CASC7竞争表达促进人精原干细胞的增殖和DNA合成，抑制其早期凋亡。6）PAK1/PDK1/miRNA-31-5p分子网络通过KDR/ZNF367与ERK1/2及AKT信号通路调控人类精原干细胞的自我更新与凋亡。7）揭示P63基因突变导致雄性生殖细胞凋亡的转录机制。8）构建三维培养体系诱导人类精原干细胞分化为有受精和发育能力的精子细胞。利用免疫磁珠分选（MACS）方法获得高纯度的人精原干细胞，通过Matrigel构建三维共培养体系，并添加多种因子和激素，将人精原干细胞体外诱导分化为单倍体精子细胞。三维诱导体系可以模拟睾丸微环境，促使人精原干细胞发生减数分裂。流式细胞分选（FACS）结果表明，诱导过程检测到了四倍体细胞和单倍体细胞。细胞染色体铺展和免疫细胞化学表明四倍体细胞为精母细胞，单倍体细胞为圆形精子细胞。荧光原位杂交(FISH)显示，人精原干细胞分化获得的精子细胞具有正常的染色体数目。单细胞RNA测序和表观亚硫酸氢盐测序结果表明，诱导获得的精子细胞具有正常的基因组特征和甲基化水平。圆形精子卵细胞浆内显微注射（ROSI）表明，诱导获得的精子细细胞具有受精和发育能力。本项目研究为人类正常精子发生机制和无精子症病因提供新的表观遗传与遗传调控机制，为男性不育的治疗提供新的靶标和功能精子细胞，对治疗男性不育与预防子代出生缺陷有重要意义。