nc886与核纤层蛋白laminA/C在前列腺癌侵袭转移中的作用及调控机制研究

The high inter-tumor heterogeneity of prostate cancer is an important feature that differents from the other tumors.The latest research suggests that lamin A/C may be a risk biomaker to estimate the prognosis of tumor; A novel type of non-coding RNA, nc886, in a wide variety of tumor may play the putative role of tumor suppressor, its deactivation by the method of the CpG methylation is a biomarker of poor prognosis in tumors. At the same time, nc886 is transcribed by RNA polymerase III (Pol III) and is the first case of a Pol III genes whose expression is regulated using the epigenetic silencing in several types of cancer. Here, our pilot study have identified that nc886 expression in human cervical cancer tissue, cell and prostate cancer cells with interstitial cell properties increased, however in the epithelium prostate cancer cells decreased; nc886 might bind laminA/C by reporter gene assay in prostate cancer cells; what role and the specific regulatory mechanism beween nc886 and laminA/C in prostate cancer have not been provided yet. We speculated that nc886 and laminA/C are putative important biomarkers in epithelial mesenchymal transition process of prostate cancer; In additon, nc886 may change the stable of the nuclear through regulating the laminA/C expression. We will investigate the role, the regulation mechanism beween nc886, laminA/C and some moleculars involving in the epithelial mesenchymal transition pathway, and the treatment by targeting nc886 in prostate cancer. Our project will focus on three aspect research to clarify the significance of abnormal expression of nc886 or laminA/C in prostate cancer progress. The first aim is to investigate the relevance between nc886, laminA/C and tumor prognosis; the second aim is to investigate the regulation mechanism and the emphasis on the relevance between nc886 and laminA/C and epithelial mesenchymal transition signal pathways; At last, our aim is to investigate the curative effect and mechanism when targeting nc886 in animal model of invasive prostate cancer. In summary, we will supply enough experimental datas to support our views for nc886 or laminA/C as a new molecular biomaker in the progression of prostate cancer and potentially novel target for invasive prostate cancer therapeutics.

前列腺癌高度异质性是其区别于其它肿瘤的重要特点。最新研究提出laminA/C可能是肿瘤预后不良的标志物；新发现的非编码RNA886（nc886）甲基化失活亦是肿瘤预后不良的标志物。前期研究识别nc886在间质性质的人类宫颈癌及前列腺癌细胞系中高表达，然而在上皮性质的前列腺癌细胞系中低表达；前列腺癌细胞系中发现nc886可以结合laminA/C，它们在前列腺癌中的角色及具体调控机制，尚无文献报道。我们推测nc886和laminA/C可能是前列腺癌上皮间质转换过程中的重要标志物；nc886可能通过调控laminA/C改变核的稳定性。本项目拟从nc886在前列腺癌中的作用、调控及治疗三个层面揭示nc886与肿瘤预后的关联性、nc886与laminA/C及上皮间质转换信号通路的关联性及靶向前列腺癌中nc886的疗效及机制，为揭示新的肿瘤标志物及治疗靶标提供可靠的实验依据。

我们前期研究很明确指出，核纤层蛋白lamin A/C与β-catenin相互作用，通过上皮间质转换（epithelial to mesenchymal transition，EMT）的机制调控癌细胞的迁移及侵袭，是识别前列腺癌分化和判断预后的新的肿瘤标志物。然而，lamin A/C是个致死性基因，不论敲除或者敲低，细胞均难以存活。在临床样品的检测中，我们发现lamin A/C的突变率很低，其表达量的高低只能在病理切片中通过免疫组化的方法检测与分析。另外，血液中的检出率极低。已在前期研究证实，nc886是可以直接调控lamin A/C表达的新型非编码RNA。因此，为了筛选更为精准的、对病人无损伤性的早期前列腺癌的标志物及治疗靶点，本项目的研究重点在于评估nc886对前列腺癌侵袭转移中的作用及对laminA/C的调控机制上。目前，已取得很多重要的预期及非预期研究结果，总结如下： ⑴ 对nc886的功能学研究中，我们明确了它在前列腺癌中的抑癌基因角色，及它在前列腺癌样品中低表达的机制。原位杂交方法发现nc886在30例前列腺癌芯片样品中低表达，与Gleason分级无明显相关性，通过103例GEO高通量测序分析验证了该结果。同时通过甲基化测序，明确了前列腺癌组织中nc886的低表达与启动子区的高甲基化密切相关； ⑵ 离体及动物实验阐明nc886显著降低了前列腺癌细胞的转移及侵袭及其机制。采用具有骨转移能力的细胞模型，稳定过表达nc886，显著引起细胞的侵袭力下降，在动物实验中观察到骨转移明显降低，非常有趣的是发现动物脾坏死减轻。在机制研究中，我们发现nc886可以直接靶向snail蛋白，直接或者间接促进snail的降解，增加E-cadherin的表达，改变癌细胞的EMT状态，降低转移。除此之外，nc886与laminA/C的表达在低Gleason分级时出现显著负相关，由此我们判断nc886亦可以通过下调laminA/C的表达，降低前列腺癌的恶性程度。肯定了nc886靶向的广泛性。同时，我们也发现转移生长因子β1（TGFβ1）可以瞬时抑制nc886的表达，干扰nc886抑制转移的效果；⑶ 我们识别出nc886在固有免疫中的重要作用，这是本项目中发现的重要非预期结果。nc886可以降低骨髓中M2型巨噬细胞的数量，进而降低骨转移，其详细机制有待阐明。