MiR-203下调Nanog表达抑制食管鳞癌上皮-间质转化及转移的机制研究

Plenty of data implicate epithelial-mesenchymal transitions (EMT) as a major factor in the migratory invasion and metastasis of epithelial cancers. Recent reports further demonstrated that miRNAs regulate EMT in several cancers including bladder carcinoma, breast cancer, lung cancer, ovary cancer and prostate cancer. However, the role of micro RNAs（miRNAs）in the regulation of EMT in esophageal squamous cell carcinoma (ESCC) remains unknown. .In a recent work we demonstrated that miR-203 significantly inhibited the migration,invasion and EMT of ESCC cells and that the expression of miR-203 was inversely correlated with lymph node metastasis in ESCC patients. In addition, we found that miR-203 significantly inhibited the expression of Nanog, which reportedly plays a key role in the maintenance of self-renewal of normal epithelial stem cells and induces EMT in lung adenocarcinoma. Furthermore, activation of Nanog was found to induce EMT of ESCC cells and re-expressing Nanog in miR-203 transfected ESCC cells significantly attenuated miR-203-induced inhibition of EMT. These data suggest that miR-203 might regulate EMT of ESCC through the Nanog-mediated signal pathway. Notably, however, bioinformatic analysis and luciferase reporter assay showed that Nanog was not a target of miR-203. Therefore, the mechanism that underlie miR-203 regulation of Nanog expression and EMT warrant further investigation..In this project, we propose to evaluate the role of miR-203/Nanog pathways in the EMT of ESCC by multiple approaches including bioinformatic analysis, luciferase reporter assay, RNA interference, ChIP, ChIP-chip, metastasis assay in NOD/SCID mice and clinical investigation. Conceivably, elucidation of the molecular pathways involved inthe regulation of EMT of ESCC by miR-203 will provide important clues for the development and evaluation of novel therapies against ESCC.

现有研究表明，上皮-间质转化（EMT）与上皮类肿瘤的转移发生密切相关。尽管近来有关miRNA调控肿瘤EMT的机制研究已取得了一定的进展，但迄今关于miRNA对食管鳞癌EMT的调控作用尚未见报道。我们在前期发现，食管鳞癌中miR-203的表达水平与EMT及侵袭转移呈显著负相关，细胞学实验提示miR-203可能通过间接下调Nanog表达抑制食管鳞癌EMT，但其具体的作用机制仍有待于进一步明确。本项目拟通过ChIP、荧光素酶报告基因实验明确调控Nanog转录的miR-203靶基因，采用ChIP-chip、生物信息学分析及细胞功能实验明确Nanog调控EMT的途径，籍此建立miR-203下调Nanog表达抑制食管鳞癌EMT的信号通路；此外，我们还将通过动物转移成瘤模型及临床统计分析评价该通路中的关键基因在食管鳞癌转移治疗和诊断中的潜在应用价值，为今后开展临床食管鳞癌转移相关的诊疗研究提供理论依据。

食管鳞癌(ESCC)是严重威胁人类健康和生存的恶性肿瘤疾病。前期研究结果显示，miR-203可以通过抑制食管鳞癌EMT进而抑制其侵袭和转移，但其在食管鳞癌中的具体调控机制尚不明确。.在本项目中，我们通过染色质免疫共沉淀、荧光素酶报告基因及相关细胞功能实验明确在食管鳞癌细胞中miR-203对NANOG表达的调控作用及具体机制；进而通过细胞功能实验探讨miR-203-NANOG途径中关键基因对食管鳞癌细胞迁移、侵袭及EMT的调控作用；此外我们还通过免疫缺陷小鼠成瘤实验和临床调查对食管鳞癌中miR-203-NANOG途径中关键基因表达水平与肿瘤侵袭转移影响的相关性作进一步的探讨。 .研究结果显示，食管鳞癌细胞中miR-203可直接抑制转录因子SP1和ZFP281的表达，而SP1及ZFP281均具有调控NANOG转录的作用，提示miR-203可能通过抑制SP1/ZFP281表达的途径间接抑制NANOG的表达。随后我们通过ChIP对NANOG下游潜在靶基因进行筛选，并通过Ad.NANOG感染食管鳞癌细胞后qPCR检测相关靶基因表达加以验证。结果显示CXCR4、EZH2、TCF3、KDMIA等基因表达显著上调，而TDGF1、SAII4、DPPA3等表达显著下调。细胞功能实验结果显示，NANOG过表达具有促进食管鳞癌细胞EMT及侵袭迁移的作用，而抑制CXCR4表达可以促断NANOG相应的促进作用。动物成瘤实验结果显示，过表达miR-203及抑制SP1、NANOG、CXCR4表达可显著抑制食管鳞癌细胞体内致瘤能力。临床病理分析结果显示，miR-203-NANOG途径中关键基因表达水平与与肿瘤的分期、分化程度及转移相关，而与患者年龄、性别等无显著相关性。.我们的研究发现miR-203可以通过抑制SP1/ZFP281等转录因子的表达的途径间接抑制NANOG表达，进而抑制食管鳞癌细胞的EMT、迁移及侵袭能力。通过动物实验及临床调查证实miR-203-NANOG途径与食管鳞癌进展相关。