长链非编码RNA CRNDE调控LATS1表达在胰腺癌细胞恶性转化中作用机制研究

Long noncoding RNA (lncRNA) is a newly discovered epigenetic regulator and plays an important role in tumor development. However, the role of lncRNA in pancreatic cancer is still lack of awareness. Our previous study found a novel lncRNA CRNDE overexpressed in pancreatic cancer by lncRNA microarray assay, and the level of CRNDE expression was closely related to LATS1 expression. LATS1 is a key member of Hippo-YAP pathway, and has methylation sites in the promoter region. Therefore, we hypothesized that CRNDE regulated LATS1 expression, and played an important role in pancreatic cancer development through Hippo-YAP pathway, but its mechanism was still not clear. This paper intended to firstly study CRNDE expression in pancreatic cancer and its clinical significance; and then to study the effects of CRNDE expression on pancreatic cancer cell proliferation, apoptosis and invasion in vitro and in vivo; finally, to reveal the molecular mechanism of CRNDE regulates LATS1 expression by methylation in the malignant transformation of pancreatic cancer cells through Hippo-YAP pathway. And these will provide a theoretical foundation to further explore the mechanism of pancreatic cancer progression and search for new therapeutic targets.

长链非编码RNA（lncRNA）作为一种新发现的表观遗传调控因子，在肿瘤的发生发展中发挥重要作用。目前对lncRNA在胰腺癌中的作用及相关分子机制还缺乏深入研究。我们前期研究通过lncRNA芯片发现一种在胰腺癌组织中高表达的CRNDE，同时还发现CRNDE的表达与LATS1分子表达水平密切相关。LATS1是Hippo-YAP通路的关键分子，而且在其启动子区含有甲基化位点。我们推测CRNDE可能通过调控LATS1表达介导Hippo-YAP通路在胰腺癌发生发展中发挥作用，但具体分子机制尚不明确。本课题首先拟明确CRNDE在胰腺癌组织中的表达及临床意义；然后在细胞与动物水平研究CRNDE对胰腺癌细胞增殖、凋亡与侵袭的影响；最后探讨CRNDE通过甲基化修饰调控LATS1表达，介导Hippo-YAP通路在胰腺癌细胞恶性转化中的分子作用机制。本研究将为深入探索胰腺癌发病机理和寻找新的治疗靶点奠定基础。

近年来，研究发现长链非编码RNA（lncRNA）通过表观遗传学的调控机制，在人类恶性肿瘤的发生发展中发挥重要作用。我们前期研究通过lncRNA芯片发现一种在胰腺癌组织中高表达的长链非编码RNA CRNDE，同时还发现lncRNA CRNDE的表达与Hippo-YAP通路中关键分子LATS1分子表达水平密切相关。因此，我们推测lncRNA CRNDE可能通过调控LATS1表达介导Hippo-YAP通路在胰腺癌发生发展中发挥作用。为了证实以上推测，首先我们在80例临床胰腺癌组织样本中证实lncRNA CRNDE在胰腺癌组织中的表达显著增高，并且结合临床资料分析证实lncRNA CRNDE的表达水平与肿瘤大小，肿瘤分化程度、TMN分期、淋巴结转移以及远处转移等临床病理特征显著相关，同时研究还发现lncRNA CRNDE可能是预测胰腺癌患者的不良预后的一个独立因素。然后，我们构建CRNDE的真核表达载体和siRNA干扰载体稳定转染入胰腺癌细胞株，结果表明下调CRNDE的表达水平可显著抑制胰腺癌细胞增殖、促进胰腺癌细胞凋亡以及减弱胰腺癌细胞在体外侵袭力，并经裸鼠移植瘤模型证实下调CRNDE的表达能够明显抑制移植瘤的生长。最后，运用去甲基化药物5-Aza-CdR干扰LAST1启动子甲基化，结果发现LAST1表达上调，Hippo-YAP信号通路激活，使得YAP1表达受到抑制，从而揭示了长链非编码RNA CRNDE是通过甲基化修饰机制抑制LAST1基因表达，致使抑制 Hippo-YAP信号通路的活化，在胰腺癌细胞恶性转化中的发挥重要的作用。本项目研究成果将为深入探索胰腺癌发病机理和寻找新的治疗靶点奠定基础。