差异表达lncRNA在伊马替尼耐药胃肠道间质瘤细胞中作用机制的研究

With a rising of morbidity and mortality rates, gastrointestinal stromal tumor(GIST) is becoming the most threaten on human health and survival. Currently, patients showed imatinib resistance, seriously affect the prognosis of patients with GIST. Due to the molecular mechanism has not been fully elucidated, it become a demanding clinical medicine problem for further study. Mounting researches suggest that long non-coding RNA plays an important role in tumor resistance. Our study established the concentration gradient of imatinib resistant cell lines for RNA sequencing, after filtered, the RNA level of RP11-616M22.7 was increased steadily along with the imatinib concentration. In vitro experiments have confirmed that RP11-616M22.7 possessed the oncogenic function in gastrointestinal stromal tumor cell lines. To explore the mechanism of RP11-616M22.7 in GIST imatinib resistance, we performed RNA Pull-down and RNA immunoprecipitation assays to explore the binding downstream effector proteins of RP11-616M22.7. After screening the candidate proteins, RASSF1 was confirmed to specifically binding with RP11-616M22.7, and then participate in the MAPK signaling pathway for tumor-promoting. Taken together, those results provide new theoretical basis for imatinib resistance of gastrointestinal stromal tumor.

胃肠道间质瘤是一种发病率和死亡率都不断上升的肿瘤，对人类的健康和生存构成了很大的危害。目前，甲磺酸伊马替尼耐药严重影响胃肠道间质瘤病人的预后，耐药的分子机制是亟待研究的问题。越来越多的研究表明长链非编码RNA在肿瘤耐药过程中起着重要的调控作用。本课题构建药物浓度梯度的伊马替尼耐药细胞株进行RNA测序，经过筛选，RP11-616M22.7的表达量随着药物浓度升高持续上调，而且体外细胞实验已经证实RP11-616M22.7在胃肠道间质瘤细胞系中作为一个癌基因功能的lncRNA发挥着重要的功能。通过RNA Pull-down和RNA 免疫共沉淀实验我们证实RP11-616M22.7可以和RASSF1蛋白相互作用，继而参与MAPK信号通路。综上研究，为胃肠道间质瘤伊马替尼耐药机制提供新的理论基础。

越来越多的证据表明，长链非编码RNA(lncRNAs)在人类癌症中发挥着重要作用。然而，对于lncRNA及其在胃肠道间质瘤(GIST)治疗中的作用一直缺乏系统的描述。我们对10个GIST肿瘤样本和配对相邻的正常样本进行了高通量RNA测序(RNA-seq)。通过生物信息学分析来表征GIST中lncRNA的转录格局和失调。我们采用5’和3’cDNA末端快速扩增(RACE)分析和亚细胞分馏来揭示RP11-616M22.7的特性。利用RNA干扰和慢病毒构建在GIST细胞中敲除并过表达RP11-616M22.7。与相邻的正常样本相比，我们在GIST样本中发现了866个上调lncRNA和1268个下调lncRNA，其中大多数是GIST特异性的，而不是其他类型的癌症。在GIST样本中发现大多数的特征是失调的，lncRNA与癌症相关的特征高度相关。与非耐伊马替尼样品相比，耐伊马替尼样品中RP11-616M22.7的水平被鉴定为升高。进一步分析发现，RP11-616M22.7与Hippo信号通路密切相关。通过用不同剂量的伊马替尼治疗GIST肿瘤细胞，我们证实了敲低RP11-616M22.7可促进肿瘤细胞的敏感性，而过表达RP11-616M22.7可诱导对伊马替尼的耐药。我们进一步证实在体内通过敲除RP11-616M22.7可以降低对伊马替尼的耐药性。此外，发现RP11-616M22.7与RASSF1蛋白相互作用。lncRNA被发现与GIST中的肿瘤相关特征密切相关。在体内和体外，RP11-616M22.7的缺失能够降低GIST肿瘤细胞对伊马替尼治疗的耐药性。