长链非编码RNA RP11-1100L3.8对非小细胞肺癌厄洛替尼耐药的机制研究

Aquired resistance to EGFR-TKI in non small cell lung carcinoma (NSCLC) greatly troubled the clinical therapy. The roles of lncRNAs in the acquired resistance of NSCLC, however, are far from being fully elucidated. Our previous work has shown that miR-451 was significantly downregulated in NSCLC tissues compared with corresponding non-tumor lung tissues. Using lncRNA chip high throughput analytical technique, we found that lnc- RP11-1100L3.8 was upregulated more than 11 times in erlotinib-acquired resistant NSCLC tissues than erlotinib-sensitive NSCLC tissues. Downregulation of lnc- RP11-1100L3.8 induced apoptosis enhancement in erlotinib-resistant NSCLC cells. Moreover, according to the competing endogenous RNA(ceRNA) hypothesis, lncRNAs may act as endogenous sponges for miRNAs for sharing miRNAs responses elements(MREs), inhibiting normal miRNA targeting activity on mRNA., thereby affecting tumor development and progression.Here, the results of luciferase reporting experiments showed that lnc- RP11-1100L3.8 could directly bind to miR-451 seed sequence. Therefore, we hypothesized that lnc- RP11-1100L3.8 could play the ceRNA activity to competitively bind to miR-451, relieving the inhibition of RAB14, thus leading to erlotinib resistance in NSCLC. We will verify the aboved hypothesis by use of clinical samples, RNA immunoprecipitation, luciferase reporter, a nude mouse model of lung erlotinib resistance and other techniques. This study will enrich the molecular mechanism of the acquired resistance of NSCLC, and provide a novel molecular target for overcoming the acquired resistance in NSCLC.

非小细胞肺癌（NSCLC）EGFR-TKI获得性耐药极大地困扰临床治疗，lncRNA与NSCLC获得性耐药的研究甚少。我们前期研究证实：miR-451在NSCLC组织中显著低表达，运用lncRNA 芯片技术发现在厄洛替尼获得性耐药的NSCLC组织中，lnc- RP11-1100L3.8表达水平显著高于敏感组织11.7 倍，敲低lnc-RP11-1100L3.8诱导耐厄洛替尼肺癌细胞的凋亡，同时荧光素酶实验证实lnc-RP11-1100L3.8与miR-451种子序列特异性结合。据此提出假设： lnc-RP11-1100L3.8通过竞争性结合miR-451，解除miR-451对靶基因RAB14的抑制，进而激活相关信号通路，导致厄洛替尼耐药。本课题以NSCLC为研究对象，拟采用荧光素酶、RIP等实验在细胞、动物及临床标本水平验证此假说，为NSCLC厄洛替尼获得性耐药的生物治疗提供新靶标及依据。

非小细胞肺癌（NSCLC）EGFR-TKI获得性耐药的机制研究是肿瘤学的研究热点。运用lncRNA 芯片技术发现在厄洛替尼获得性耐药的NSCLC组织中，lnc- RP11-1100L3.8表达水平显著高于敏感组织约11.7 倍.在收集临床病例的过程中，我们还发现了两例接受了EGFR-TKI的药物治疗晚期NSCLC伴肠转移的病例，通过这两例患者，我们观察到晚期NSCLC患者给予EGFR-TKI治疗后不同的临床获益和预后，而其内在的机制需要进一步更深层次的研究。在细胞水平，项目组经过研究证实，在耐厄洛替尼的NSCLC细胞株PC9/TR、HCC827/TR中lnc- RP11-1100L3.8的表达水平较敏感细胞中表达显著上调，敲低lnc- RP11-1100L3.8后，MTT实验显示细胞增殖活性显著下降，qRT-PCR检测显示miR-451表达水平明显上调，流式细胞分析显示细胞凋亡明显增加。过表达lnc- RP11-1100L3.8后，细胞增殖活性显著上升。荧光素酶报告实验证实了lnc- RP11-1100L3.8与miR-451种子序列特异性结合。在PC9/TR、HCC827/TR中，过表达lnc- RP11-1100L3.8后转染miR-451的过表达载体，在细胞水平观察发现上述拯救实验可逆转因lnc- RP11-1100L3.8过表达对细胞厄洛替尼敏感性的影响。本课题以NSCLC为研究对象，采用荧光素酶、MTT、流式细胞分析等实验获得了lnc- RP11-1100L3.8调控miR-451增强非小细胞肺癌厄洛替尼耐药性的功能学依据，为NSCLC厄洛替尼获得性耐药的生物治疗提供新靶标及依据。