EZH2调控长链非编码RNA SPRY4-IT1促进NSCLC细胞侵袭转移的机制研究

Multiple lines of evidences link dysregulations of long noncoding RNAs (lncRNAs) to diverse human diseases, especially cancers. The roles of lncRNAs in the carcinogenesis of non-small cell lung cancer (NSCLC), however, are far from being fully elucidated. Our previous work has shown that down-regulated lnc SPRY4-IT1 was obviously associated with advanced pathological stage,lymph-node metastasis, and shorter overall survival of NSCLC patients. Over-expression of lnc SPRY4-IT1 in NSCLC cells was demonstrated to decrease the cell proliferation, migration, invasion, while lead to the promotion of cell apoptosis in vitro. Meanwhile, we found that the histone H3 lysine 27 (H3K27) tri-methylating enzyme, Enhancer of zeste homolog 2 (EZH2) may mediate silencing expression of lnc SPRY4-IT1. Recent studies show that lncRNAs have typical histone modifications that are involved in cancer processes. Moreover, according to the ceRNA (competing endogenous RNA) hypothesis, lncRNAs may elicit their biological activity through their ability to act as endogenous sponges for miRNAs, and such activity could in turn affect the distribution of miRNAs on their targets, thereby affecting tumor development and progression. Here, the results of luciferase reporting experiments showed that lnc SPRY4-IT1 could directly bind to miR-197/23a seed sequence. Therefore, we suppose that lnc SPRY4-IT1 may be a crucial metastatic factor, and silenced by EZH2 mediated epigenetic regulation. It could play the ceRNA activity to regulate miR-197/miR-23a function, thus leading to the activation of tumor invasion and metastasis-related signaling pathways. We will identify the aboved hypothesis by use of clinical samples,Chromatin immunoprecipitation, luciferase reporter , a nude mouse model of lung metastasis and other techniques. This study will enrich the molecular mechanism of NSCLC invasion and metastasis, and provide a novel molecular target for diagnosis, treatment and prognosis of advanced NSCLC.

LncRNA与非小细胞肺癌（NSCLC）发生发展的研究报道甚少。我们前期工作表明：组蛋白甲基转移酶EZH2能调控lnc SPRY4-IT1表达； 且lnc SPRY4-IT1表达下调，与NSCLC患者TNM分期、淋巴结转移及预后差密切相关；体外实验证实lnc SPRY4-IT1可影响NSCLC细胞的迁移侵袭能力，其分子机制值得深入研究。文献表明lncRNA的转录亦受组蛋白修饰调控，且lncRNA可作为 ceRNA通过miRNA调控肿瘤进程。本课题组已证实lnc SPRY4-IT1能与miR-197/23a种子序列直接结合，据此提出假设：EZH2介导表观遗传学水平沉默lncRNA SPRY4-IT1的表达，减弱其调控miR-197/23a的功能，从而促进肿瘤侵袭转移。拟通过临床标本，ChIP、荧光素酶实验、裸鼠模型等方法证实上述假设，为NSCLC发病机制，临床诊断、治疗及预后提供新理论依据。

背景：新近越来越多的长链非编码RNA(LncRNAs)被鉴定，表明lncRNAs在癌症发病过程中发挥重要作用,并可能为这种疾病生物学发生提供新的见解。本课题旨在研究lncRNA SPRY4-IT1在非小细胞肺癌（NSCLC）组织及细胞中的表达水平对细胞侵袭转移功能的影响及其异常表达的分子机制。.内容：定量PCR检测了SPRY4-IT1在NSCLC组织中的表达，并分析了其表达水平与病人临床特征的相关性。ChIP方法检测EZH2对SPRY4-IT1的调控。划痕，transwell和活体动物实验检测了SPRY4-IT1对细胞迁移和侵袭转移能力的影响。Western blot，免疫荧光检测了SPRY4-IT1对下游靶基因的调控。.结果：SPRY4-IT1在NSCLC组织及细胞中表达下调，并与NSCLC病人的预后差密切相关，且SPRY4-IT1可能受到组蛋白甲基化转移酶EZH2的调控。NSCLC细胞中过表达SPRY4-IT1能抑制细胞增殖、侵袭转移能力；干扰SPRY4-IT1的表达则能促进细胞的侵袭转移能力。Western blot及免疫荧光结果显示SPRY4-IT1可能是通过调控NSCLC细胞的上皮-间质转化（EMT）影响细胞的增殖和侵袭转移能力。NSCLC细胞中封闭EZH2的表达同样能抑制细胞增殖和侵袭转移能力，与过表达SPRY4-IT1的结果一致。拯救实验证实SPRY4-IT1是EZH2发挥癌基因的一个重要的下游因子。.意义：EZH2通常在非小细胞肺癌组织中表达，其表达水平与鳞状细胞癌的早期发病机制和肺腺癌的进程相关。EMT作为机体分化过程中的一个重要程序，参与胚胎发育早期阶段的组织重构,并与肿瘤细胞侵袭和转移相关。本研究提示在NSCLC中EZH2抑制lncRNA SPRY4-IT1的转录表达，SPRY4-IT1作为EMT的调控因子参与NSCLC细胞的侵袭转移过程，并可能作为NSCLC病人预后差的分子标记物。