长链非编码RNA在人卵巢早衰发生中的作用机理及分子机制研究

Premature ovarian failure (POF) is a common female disease which severely affect female fertility and life quality, however, the reason for the cause of such disease now is poorly understood, especially the lack of long non-coding RNA (lncRNA) in the regulation of human POF. We previously found that the expression profile of lncRNAs between ovarian tissues from patients with POF and normal fertility were significantly different, and these differentially expressed lncRNAs were mostly highly expressed in female reproductive systems, such as oviduct tissues, granulosa cells and ovarian tissues. In addition, some associated factors related to the regulation of granulosa cell proliferation was also affect the expression of some of these differentially expressed lncRNAs. Therefore, these results may indicate that these differentialy expressed lncRNAs be involved in the occurrence of human POF, but the specific mechanisms are still not understood. Thus, we propose the hypothesis that the lncRNA may regulate the biological function of granulosa cells, such as proliferation, then leading to the occurrence of POF. In order to validate this hypothesis, materials of human granulosa cell line KGN, primary granulosa cell and ovarian tissue were used to investigate the important role of lncRNA in the regulation of human POF and its molecular mechanisms by using bioinformatical analysis, real-time PCR, western blot, RNA interference, flow cytometry, RNA fluorescence in situ hybridyzation and RNA immunoprecipitation, through levels of molecular, cellular and histological. This study will provide a new vision for the foundations of uncovering the important role lncRNA in the occurrence mechanism of POF, and provide a new way to diagnosis and treatment of female POF and its complications based upon the aspects of lncRNA.

卵巢早衰严重影响女性生育和生活质量，目前对其发生的分子机制知之甚少，尤其是长链非编码RNA（lncRNA）调控卵巢早衰发生的研究尚未见报道。我们前期发现早衰卵巢组织lncRNAs表达谱异常，且这些lncRNAs多高表达于女性生殖系统中，调控颗粒细胞增殖的相关因素可影响此类lncRNAs的表达，提示lncRNAs可能参与卵巢早衰的发生。为此，我们提出假说：lncRNA 可能通过影响颗粒细胞增殖等作用导致卵巢早衰。为验证此假说，我们将通过人颗粒细胞系KGN、原代颗粒细胞和卵巢组织，采用生物信息学分析、RNA干扰、流式细胞术、RNA荧光原位杂交、RNA免疫共沉淀等手段，从分子、细胞及组织水平等多方面探讨lncRNA在卵巢早衰发生中的重要作用及其分子调控机制。本研究将从lncRNA这个新视角为揭示卵巢早衰发病机制奠定基础，并为全新的基于lncRNA的诊断和治疗女性卵巢功能衰退及并发症提供新的思路。

卵巢早衰（POF）严重影响女性健康，一方面导致不孕，另一方面由于雌激素分泌不足导致如骨质疏松等女性相关疾病。导致POF发生的大多数病因不清，也无有效治疗方案。近年来的研究发现长链非编码RNA（lncRNA）是一类具有疾病和组织特异性的非编码RNA。本研究通过对POF和正常患者的卵巢皮质组织进行二代测序分析，发现多个差异表达的lncRNA和mRNA，通过进一步筛选差异表达lncRNA和mRNA，从细胞、组织等层面分析其潜在的调控关系以及在调控卵巢功能中的重要作用。结果发现20个显著差异表达的lncRNAs（包括12个上调和8个下调）、52个差异表达的mRNAs（包括33个上调和19个下调）。通过GO和KEGG分析发现差异表达转录物与卵泡发育和颗粒细胞功能密切相关。此外，有13个差异表达lncRNAs及其靶向的mRNAs在卵巢皮质组织中存在共表达关系，包括lnc-ADAMTS1-1:1/ADAMTS1、lnc-PHLDA3-3:2/CSRP1、lnc-COL1A1-5:1/COL1A1、lnc-SAMD14-5:3/COL1A1和lnc-GULP1-2:1/COL3A1。通过检测差异表达lncRNAs在患者血清中表达情况，结果发现可以在血清中稳定检测到其表达，并且表达水平与卵巢组织中的结果一致。进一步的分析发现lnc-GULP1-2:1和COL3A1在卵巢皮质组织中均显著低表达，并且生物信息学的分析结果表明COL3A1是lnc-GULP1-2:1的潜在靶标。通过构建lnc-GULP1-2:1的KGN稳转过表达细胞系，结果表明lnc-GULP1-2:1可以显著促进COL3A1的表达；免疫荧光研究发现lnc-GULP1-2:1不仅影响了COL3A1的表达水平，也影响了COL3A1的亚细胞定位。这些结果表明POF患者卵巢组织和血清样本中具有差异表达的关键lncRNAs的存在，对于此类lncRNAs的深入研究为阐明POF的发病原因来说至关重要。对血清样本差异lncRNA的表达分析将会为POF的早期诊断和治疗提供依据。