长链非编码RNA plzfrl在精原干细胞分化过程中功能和机制探讨

Long non-coding RNAs (lncRNAs) are involved in stem cell differentiation, and organ development. Spermatogonial stem cells（SSCs）are the foundation of spermatogenesis. We have reported a novel SSC self-renewal associated lncRNA, moreover, we also identified another SSC differentiation associated lncRNA through previous high throughput sequencing study. Interestingly, this latter lncRNA, called lncRNA plzfrl was transcribed from the antisense region of intron 4 of plzf gene and there was a clear concomitant change of the transcript levels of lncRNA plzfrl and plzf in SSCs. PLZF has been reported as a key transcription factor regulating SSC stemness. Our preliminary data indicated this lncRNA was specifically expressed in mouse spermatogonia and responsive to retinoic acid signal, suggesting lncRNA plzfrl is involved in SSC differentiation. We continue to analysis its roles in SSCs by silence or activation of lncRNA plzfrl, together with functional transplantation. We further conduct CHIRP(Chromatin Isolation by RNA Purification) and RNA-pull down experiments to underlie its possible molecular mechanism. Our study might contribute novel clues for differentiation regulation of male germline stem cells.

长链非编码RNA在干细胞分化和器官发育过程具有重要作用。精原干细胞是生精的基础，最近我们报道了一个长链非编码RNA（lncRNA）参与精原干细胞的自我更新，此外我们从深度测序结果还找到了一个lncRNA和SSC分化调控潜在相关。令人感兴趣的是，这个4.6kb长的lncRNA起源于Plzf内含子4反义序列区域，且与Plzf共表达，而PLZF则是维持SSCs干性的关键转录因子。LncRNA plzfrl在精原细胞中特异表达，受因子维甲酸调控，提示它参与调节SSCs的分化。本项目拟通过表达沉默及过表达，结合体内功能移植实验分析其功能，及CHIRP和RNA-pull down技术，阐明其可能分子机制，为研究雄性干细胞的分化调控提供新的线索。

作为一类具有生物学功能的非编码RNA的增强子RNA(eRNAs)，在组织和细胞发育的基因调控中发挥作用。精子发生是雄性遗传物质传播的基础，而精子发生中的eRNA表达谱尚未揭示，功能调控作用尚未报道。我们通过重力沉降法(STA-PUT)分选与磁珠分选结合，收获小鼠睾丸不同类型的生精细胞，使用这些细胞，通过CHIP-seq和RNA-seq方法分析精子发生过程中eRNA表达的全局概况。eRNA数量在不同生精细胞中呈现先降低再升高的趋势，其中精母细胞中eRNA数量最少，提示eRNA具有细胞特异性，而精原细胞中的eRNA分布在基因间的数目显著高于其他两类细胞，提示精原细胞中增强子eRNA的作用与其他两类有所不同。我们进一步利用双荧光报告素酶系统筛选得到了Tdrd1上游的eRNA 表达区域的DNA片段具有促进Tdrd1基因表达的作用，此片段区域转录的eRNA是睾丸富集、精母细胞高表达的增强子RNA，且其在精原干细胞分化过程中表达增加，具有调控Tdrd1 mRNA表达的作用。我们的研究结果为遗传物质稳定传递提供了新的依据，为阐明精子发生的复杂调节网络提供了新线索。