lncRNA LOC554202调控miR-31介导的非小细胞肺癌EGFR-TKI耐药机制的研究

Recent evidence has indicated that re-activations of PI3K/AKT and MEK/ERK pathways are considered as two crucial mechanisms leading to EGFR-TKI acquired resistance. In our previous study，clinical and in vitro study revealed that overexpression of miR-31 and its host gene lncRNA LOC554202 were correlated with acquired resistance to EGFR-TKI. Reduced them could regain the sensitivity to EGFR-TKI in the resistant cell lines and miR-31 could regulate the resistance through PI3K/AKT and MEK/ERK pathways. Bioinformatics indicated the transcription factors regulation and epigenetic modification in LOC554202-associated promoter may contribute to transcriptional regulation of miR-31，thereby affect its expression. Here，we propose a hypothesis that transcription factors deregulation and epigenetic alterations following EGFR-TKI treatment may increased the level of LOC554202 and miR-31， and then could activate PI3K/AKT and MEK/ERK pathways， finally resulted in acquired resistance to EGFR TKI of NSCLC. The objectives of the present project are to explore the potential role of LOC554202/miR-31 in EGFR-TKI resistance of NSCLC. We will verify our hypothesis by using in vivo and vitro study as well as examination of clinical tumor tissue and circulating samples. In summary, our study may provide novel predictive biomarkers to monitor clinical response and optimize treatment strategies to overcome EGFR-TKI resistance in NSCLC patients.

PI3K/AKT和MEK/ERK重新激活是非小细胞肺癌(NSCLC)EGFR-TKI获得性耐药的关键因素。我们前期检测临床样本和细胞发现高表达的miR-31及其宿主基因lncRNA LOC554202与NSCLC TKI耐药形成相关; 细胞中敲低它们可减轻耐药，且miR-31可调控PI3K/AKT、MEK/ERK的关键因子。生物信息和文献提示LOC554202启动子区存在转录因子和甲基化调控，进而调节miR-31的转录和表达。故推测TKI治疗中，转录因子和甲基化的改变导致LOC554202上调，从而上调miR-31，进而影响miR-31下游靶通路介导了TKI耐药。本课题拟用临床样本检测和体内外功能实验，阐述LOC554202通过调控miR-31介导的通路在NSCLC TKI耐药中的作用机制，为基于LOC554202/miR-31的干预耐药策略及寻找TKI耐药相关新标记物提供理论和实验依据。

表皮生长因子受体（EGFR）突变的非小细胞肺癌（NSCLC）患者不可避免地会因EGFR酪氨酸激酶抑制剂（TKI）获得性耐药的发生而导致治疗失败。然而，关于NSCLC患者TKI耐药的机制复杂、预测标记物未明确、逆转策略有限。立足此问题，本研究在临床TKI耐药的患者进行了全面的临床特征和基因检测的分析，进一步了解了肺癌TKI耐药的临床特征和分子表型特点。近年来证据提示非编码RNA在肿瘤的耐药形成中发挥了重要作用。本研究运用体内外功能实验以及临床样本分析,阐明了miR-31和其宿主长链非编码RNA LOC554202在NSCLC TKI获得性耐药中的高表达模式以及其生物学功能和机制。进而我们还运用文献挖掘和生信分析对miR-31在恶性肿瘤中的作用及其机制进行梳理。我们证实了高表达的LOC554202上调了miR-31的表达，并且高表达的LOC554202和miR-31促进了NSCLC细胞的增殖和克隆生长，增加了TKI的药物抵抗性。TKI耐药细胞中敲低它们，则增加NSCLC细胞对TKI的敏感性；而TKI敏感细胞中过表达它们，则降低细胞对TKI的敏感性。进而我们在构建的模式动物中验证了LOC554202和miR-31对NSCLC TKI耐药的促进作用，干扰它们可以显着抑制体内对TKI耐药的NSCLC细胞的生长。进而，我们对临床TKI耐药的NSCLC患者的样本进行动态检测，证实患者耐药后的LOC554202和miR-31的表达水平较前显着升高，且LOC554202的表达与miR-31的表达呈正相关。分子机制探讨上，我们通过荧光素酶报告基因分析，确定了RAS P21蛋白激活剂1（RASA1）和缺氧诱导因子1亚基α抑制剂（FIH-1）作为miR-31在NSCLC细胞中的直接靶标。我们也证实了miR-31通过直接抑制RASA1和FIH-1的表达，至少部分激活了NSCLC中的RAF-MEK-ERK和PI3K-AKT信号通路，最终导致了TKI的获得性耐药。这些结果将为基于LOC554202/miR-31的干预耐药策略及寻找新标记物提供理论和实验依据。