lnc-LOCG110004584在非小细胞肺癌EMT及侵袭转移过程中的作用与机制研究

A continuing problem of non-small cell lung cancer (NSCLC) tumorigenesis is the metastasis of cancer cells, which is the main cause of death in patients, since epithelial-mesenchymal transition (EMT) plays vital roles during the process. Recent studies have highlighted the importance of long noncoding RNAs (lncRNAs) in the regulation of EMT and metastasis of NSCLC. Our previous work has shown that a total of 36 lncRNAs were differentially expressed at least 5.0-fold change between low invasive NSCLC samples (without visceral metastasis) and high invasive NSCLC tissues (bone/liver/brain metastasis) by lncRNA microarray analysis. Bioinformatics analysis of lncRNA sequences revealed the presence of miR-224 binding sites on lnc-LOCG110004584 which was downregulated about 9.2-fold in high invasive NSCLC tissues, as we have confirmed that miR-224 could promote epithelial phenotype of NSCLC cells under the support of previous National Natural Science Foundation of China (No.81272601). The expression of lnc-LOCG110004584 using quantitative real-time PCR (qRT-PCR) assay which performed in NSCLC tissues and cells with different invasive properties show coherence with the microarray data. Further studies found that over-expression of lnc-LOCG110004584 can decrease miR-224 while increase E-cadherin expression, and leads to surpression of invasion and migration of NSCLC cells. Otherwise, the luciferase assay confirmed that lnc-LOCG110004584 can directly bind to miR-224 through recognition sites. Therefore, we suppose that lnc-LOCG110004584 may be a crucial metastatic factor which could play the ceRNA activity to regulate miR-224/p21WAF1/CIP1 function, thus leading to activation of EMT, and then promote NSCLC invasion and metastasis. We will identify the aboved hypothesis by use of clinical samples, luciferase reporter, a nude mouse model of lung and liver 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.

我们前期通过lncRNA芯片、qRT-PCR及生物信息学等方法发现在高侵袭转移NSCLC中显著低表达的lnc-LOCG110004584的序列中含有miR-224（已证实miR-224能够促进NSCLC细胞的EMT表型形成）种子序列的结合位点。进一步研究发现过表达lnc-LOCG110004584能够下调miR-224，上调E-cadherin，并导致NSCLC细胞侵袭和迁移能力减弱；荧光素酶报告基因实验证实lnc-LOCG110004584能与miR-224种子序列结合。我们拟通过RNA干扰及过表达等实验方法在体内外水平证实lnc-LOCG110004584发挥ceRNA活性吸附miR-224实现对其靶基因p21WAF1/CIP1的调控，诱导EMT发生进而促进NSCLC侵袭转移的科学假设，从新的角度揭示NSCLC侵袭转移机制，为NSCLC诊断、治疗及预后提供新的理论依据和靶标。

肺癌是世界上最常见的恶性肿瘤之一，已成为癌症相关性死亡的头号杀手。lncRNA作为ncRNA的一个重要类型，通过多个层面调控基因表达从而参与包括肿瘤在内的多种疾病的发生、发展。我们通过体内外实验研究发现，相较于癌旁正常组织，lnc-KIAA1731-2在NSCLC中高表达，抑制其表达可增强细胞凋亡、减弱细胞侵袭转移及增殖能力；分子机制研究结果表明lnc-KIAA1731-2可能通过协同调控下游靶基因C11orf54发挥其功能。另外，根据前期研究基础及课题设想，对miR-224的功能及其作用机制进行了相关研究并发现，相较于低转移NSCLC组织和细胞，miR-224在高侵袭转移NSCLC组织和细胞中表达水平显著增高；并且，miR-224可能通过介导EMT进程影响NSCLC侵袭转移、凋亡及增殖。