长链非编码RNA(Lnc-PolE1)和DNA聚合酶Polε的表达相关性及在颈椎间盘退变中的机理研究

Telomere length is tightly associated with human aging process. However, it is still obscure about the role of telomere length in cervical disc degeneration. Our primary results revealed that the protein and mRNA level of DNA polymerase eplison (Polε) shows positive correlation with extent of cervical disc degeneration and telomere length. Clinical samples with seriously cervical disc degeneration commonly have lower Polε level and shorter telomere length. We also down-regulate Polε level in nucleus pulposus and osteoblast cells by shRNA, which can induce apoptosis and result in telomere shortening. By collecting clinical samples with seriously cervical disc degeneration and subjecting their RNAs for RNA-Seq, we identified a long non-coding RNA (Lnc-PolE1), which is highly expressed in these samples. Therefore, we speculated that Lnc-PolE1 negatively regulates Polε expression, thereby controlling telomere length and causing cervical disc degeneration. In this study, we propose to further examine more clinical samples, and down-regulate or up-regulate expression of Polε or Lnc-PolE1 in human osteoblast and nucleus pulposus cells. Based on these studies, we propose to clarify the upstream and downstream mechanisms of Lnc-PolE1 regulation and understand their roles in cervical disc degeneration. Collectively, this study will help us reveal pathogenic mechanism of cervical disc degeneration, and provide new views for its prevention and cure.

端粒长度与人类衰老过程密切相关，迄今对端粒长度与颈椎间盘退变的关系知之甚少。本课题组研究发现颈椎间盘退变越重，端粒DNA复制所需的DNA聚合酶Polε表达量越低，端粒长度越短。进一步的体外实验发现敲低Polε会诱导细胞凋亡和使端粒长度变短。通过RNA测序我们还发现一条与Polε匹配的新长链非编码RNA(Lnc-PolE1)，其在退变严重的颈椎间盘髓核细胞中高表达。因此我们推测Lnc-PolE1通过负调控Polε的表达而控制端粒长度，导致颈椎间盘退变。本项目拟扩大样本量，进一步验证Polε和Lnc-PolE1的表达量和端粒长度以及颈椎间盘退变程度的关系；通过在成骨细胞和髓核细胞中分别敲低和过表达Polε或Lnc-PolE1，明确Lnc-PolE1上下游调控机理及其在颈椎间盘退变中的作用。本研究将从Lnc-PolE1这一新视点，为揭示颈椎间盘退变的发生机制奠定基础，为其防治提供新思路。

颈椎退行性病变是机体衰老的一个重要特征，近年来，大量的研究表明端粒长度能用作衡量细胞衰老的一个极其重要的指标。但是国际国内均没有研究显示端粒DNA的复制以及其长度变化与颈椎间盘退变（IDD）过程存在相关性。本项目通过对大量的临床样本检测发现IDD患者中贫血（即缺铁）与IDD的退变程度呈显著正相关。通过对IDD样本进行microarray分析，我们发现一个含铁基因PolE的表达显著下调，而一些凋亡蛋白编码基因如Caspase-3和Caspase-8表达则上调。通过在体外细胞实验中添加铁螯合剂DFO或在髓核细胞中敲低铁转运蛋白DMT1或转铁蛋白受体蛋白TfR1的表达，我们发现PolE复杂成员的蛋白质水平显著降低，而细胞凋亡途径被激活。我们通过microarray发现在一个长链非编码RNA LncPolE会负向节剂PolE的表达。LncPolE在IDD临床样本中表达水平显著升高且与IDD的退变程度呈负相关。体外实验中过表达LncPolE会降低PolE1水平并引起细胞凋亡，在培养基中添加或者消耗铁都会影响LncPolE的表达。此外，我们发现LncPolE启动子区域DNA甲基化的下调是导致其过表达的根本原因。由于PolE对于端粒DNA的复制至关重要，因此我们也测定IDD患者的相对长度的变化，结果发现端粒长度与IDD退变程度呈负相关，即端粒越短，退变程度越严重。在细胞水平通过过表达LncPolE会下调PolE，导致端粒长度变短和引起细胞凋亡。综上所述，本研究深入阐释了LncPolE启动子区域DNA甲基化的改变如何影响LncPolE和PolE的表达，进而改变端粒长度和引起细胞凋亡，我们的结果将为IDD临床治疗提供了新的可能。