LncRNA AFAP1-AS1介导肺腺癌厄洛替尼耐药的机制研究

Currently EGFR tyrosine kinase inhibitors(TKIs) resistance has become the bottleneck of molecularly targeted therapy in Lung adenocarcinoma。The resistance mechanisms are elusive. Multifactor-regulated PTEN loss contributes to erlotinib resistance in EGFR-mutant lung adenocarcinoma by activation of PI3K/AKT/mTOR signaling. Among them, the molecular mechanisms underlying the regulatory functions of lncRNAs are still poorly understood. In our previous study, aberrantly high expression of AFAP1-AS1 was observed to be involved in erlotinib resistance in lung adenocarcinoma. The differential expressions of AFAP1-AS1 could affect erlotinib sensitivity in cancer cells. SiRNA/AFAP1-AS1 could upregulate PTEN and induce molecular alterations underlying downstream signaling. Rescue experiments suggested erlotinib sensitivity decrease could be partially abrogated by the resulting PTEN overexpression. The downregulation of PTEN is related to the catalytic activity of EZH2, a core subunit of AFAP1-AS1-bound PRC2. So, we hypothesized that PRC2-binding AFAP1-AS1 could reduce the expression of PTEN through epigenetic regulation mechanism, in turn, affect the downstream signaling pathways and consequently modulate erlotinib resistance in lung adenocarcinoma. We will confirm our hypothesis by experiments such as RIP and ChIP including assays at different levels in cells, animal, tissues. Thus, new molecular mechanisms of TKIs resistance could be found. Furthermore, either new biomarkers or new therapeutic targets could occur with translation of these findings.

耐药问题是EGFR突变肺腺癌TKIs治疗的瓶颈，其耐药机制亟待深入研究。有报道在多因素调控下PTEN缺失能够激活PI3K/AKT/mTOR信号通路引起TKIs耐药，其中lncRNAs的调控作用及分子机制还不清楚。我们预实验显示：lncRNA AFAP1-AS1在肺腺癌厄洛替尼耐药过程中异常高表达；其差异性表达与厄洛替尼敏感性相关；敲降AFAP1-AS1能够上调耐药细胞中的PTEN进而影响其下游分子表达；PTEN拯救实验能够恢复厄洛替尼敏感性；PTEN下调与AFAP1-AS1绑定PRC2蛋白复合体的主要催化亚基EZH2有关。我们推测：肺腺癌AFAP1-AS1异常高表达通过表观遗传沉默PTEN表达，继而影响下游分子信号通路调控厄洛替尼耐药。本课题拟通过在细胞、活体动物、临床组织水平的验证，并运用RIP、CHIP等实验技术证实以上推测，以丰富肺腺癌TKIs耐药机制研究，增加新标志物及治疗新靶点。

研究背景：.非小细胞肺癌（non-small cell lung cancer, NSCLC）的预后不容乐观，其发生机制仍不清楚。近年来，研究显示长链非编码RNA (Long non-coding RNA,  lncRNA)参与了许多生物学过程，在肿瘤中紊乱表达，广泛参与了其发生和发展，是肿瘤研究的新热点。进一步了解NSCLC中功能性lncRNAs的具体作用及其分子机制对于理解NSCLC的发展以及治疗靶点的寻找是十分有必要的。.主要研究内容：.我们通过qPCR探究lncRNA AFAP1-AS1在NSCLC组织中的表达量情况，以及关联病人预后和临床资料进行了分析。又通过体内、外实验探究了AFAP1-AS1的具体生物学作用。我们通过高通量测序和实验在表观调控方面探究了AFAP1-AS1发挥作用的具体分子机制。.重要结果：.本项目完成了以下主要技术指标：1、揭示并阐明了AFAP1-AS1过表达与NSCLC临床病理特征及恶性生物学表型相关；2、揭示了AFAP1-AS1可能介导NSCLC恶性表型及临床病理特征的生物学机制。同时项目在另一项研究中揭示了LINC00551在肺腺癌中低表达并可抑制肺腺癌恶性进展及调控肿瘤糖代谢重塑；还揭示了LINC00551调控肿瘤糖代谢的可能机制。.关键数据： .1. AFAP1-AS1在NSCLC组织中表达量升高并且与NSCLC患者的不良预后相关。2. 下调AFAP1-AS1的表达可以在体内和体外两个维度抑制肿瘤的增殖。3. 机制研究发现AFAP1- AS1是通过结合转录因子EZH2并将其募集到P21启动子区从而在表观水平抑制P21的表达来发挥生物学作用。4、LINC00551在肺腺癌中表达下调。5、体内、外过表达LINC00551可以抑制肺腺癌恶性进展。6、过表达LINC00551可以通过降低c-Myc转录促进PKM2表达的作用抑制肺腺癌糖酵解；同时过表达LINC00551和c-Myc可以回复PKM2的表达和糖酵解表型，从而抑制肺腺癌进展。.科学意义：.在NSCLC中差异表达的lncRNA AFAP1-AS1可能成为病人预后判断的新的分子标志物和潜在的治疗靶点。lncRNA LINC00551也有望成为肺腺癌新的生物诊断标志物和治疗靶点。