LINC00657介导内质网应激反应活化参与黑色素瘤侵袭转移的功能及机制研究

In our previous lncRNA chip experiment on screening long non-coding RNAs, we found LINC00657 was strongly related to melanocyte stress, with elevated expression in melanoma tissue and high aggressive melanoma cells. Preliminary study showed LINC00657 contributed to migration and invasion of melanoma cells by activating endoplasmic reticulum stress (ERS). And then, combined with application of ERS inhibitors and activators, we aim at proving the metastasis phenotype promoted by LINC00657 through activating endoplasmic reticulum stress in cell and animal models. Based on that, we will monitor the signaling pathways related to ERS activation of this lncRNA through the whole genome chip technology. On the other hand, we will explore the specific target genes regulated by LINC00657 through target molecular prediction software, differential gene expression profile, RNA ChIP, and dual luciferase reporter assay. Further more, by designing gene functional recovery experiment, we will confirm the possible target genes and signaling pathways involved in activation of ERS caused by LINC00657. Finally, we go from bench to bed, and detect the LINC00657, LINC00657 related target genes and key target molecules of signaling pathways expression in samples of patients tissue and blood. By doing so, we believe that some of them can be reliable bio-marker and benefit in melanoma screening, diagnosis, prognosis, or even provide new treating targets.

前期lncRNA芯片筛出LINC00657与黑色素细胞应激相关，并在黑色素瘤组织及高侵袭力黑色素瘤细胞中高表达。预实验发现LINC00657可激活内质网应激反应（ERS）进而促进A375的迁移侵袭。本课题首先将结合应用ERS抑制剂和激活剂，从细胞及动物水平验证此lncRNA介导ERS活化参与黑色素瘤的侵袭转移相关表型；在此基础上，一方面通过全基因芯片技术，筛查并验证LINC00657介导ERS活化的信号通路，另一方面利用lncRNA靶分子预测软件、差异基因筛选、RNA ChIP、荧光素酶报告分析等方法，探索LINC00657特异性调控的靶基因，再通过基因功能恢复实验，确定LINC00657介导ERS活化参与黑色素瘤侵袭转移的具体机制；最后回到临床，探索LINC00657、靶基因、信号通路关键靶分子在组织及血液标本中的表达情况，筛选出黑色素瘤可能的治疗靶点及早期诊断和预后监测的指标。

我们的研究成果明确了LINC00657在黑色素瘤组织及细胞中高度过表达，沉默LINC00657能显著降低黑色素瘤的侵袭转移能力并提高miR-205的表达，miR-205和LINC00657在RNA诱导的沉默复合体中存在相互作用。下调LINC00657几乎完全逆转了敲低miR-205所诱导的恶性黑素瘤细胞迁移和侵袭。此外，沉默LINC00657显著逆转敲低miR-205增加的EGLN2和ER相关基因水平。裸鼠成瘤实验表明下调miR-205显著增加黑色素瘤增殖、转移的能力，而下调的LINC00657很大程度上逆转了miR-205缺失的抑制作用。证实LINC00657通过海绵吸附短链非编码RNA miR-205，激活EGLN2介导的内质网应激反应活化促进肿瘤早期转移，首次阐明了黑色素瘤中LncRNA作为细胞应激调控子影响肿瘤转移的新机制。通过二代芯片及CHIRP芯片发现LINC00657可能通过调节ARF1的活性影响下游目的基因TM9SF4基因的活性，从而影响黑色素瘤的侵袭转移等生物学行为，现有的数据表明LINC00657参与黑色素瘤的发生发展，启示其可能是黑色素瘤治疗的一种新靶点。