c-Myc诱导的长链非编码RNA AFAP1-AS1促进NSCLC细胞增殖机制的初步研究

The molecular mechanism of non-small cell lung cancer (NSCLC) development is hot and difficult in the present. Recent studies have shown that lncRNAs play an important role in the process of tumor formation, one of their mechanisms is associating with chromatin-modifying complexes and affect gene expression. In our previous study, we have found that long noncoding RNA (lncRNA) lnc-AFAP1-AS1 is obviously upregulated in NSCLC tissues and correlated with clinic-pathological characteristics of patients. Interference of lnc-AFAP1-AS1 could significantly decrease NSCLC cell proliferation, migration and increase cell apoptosis. Previous studies have reported that the oncogene c-Myc could bind to the promoter region of many genes, including both coding and noncoding RNAs, and induce their expression. In previous study, we have verified that c-Myc plasmid could induce the expression of lnc-AFAP1-AS1 in NSCLC cells. It is also reported that c-Myc could increase the level of PRC2 complex family members-EZH2 and SUZ12 in stem cells. Interference of either lnc-AFAP1-AS1 or EZH2, SUZ12 could increase the expression of downstream genes. So, we hypothesised that the upregulation of oncogene c-Myc could increase the expression of lnc-AFAP1-AS1 and PRC2 complex family. lnc-AFAP1-AS1 bound to EZH2, SUZ12 and mediated transcriptional silencing of downstream tumor suppressor genes, which could be involved in the development of NSCLC. We will verify our hypothesis by using ChIP、RIP and nude mice et.al, and detecting lnc-AFAP1-AS1 expression in a large number of clinical samples in our future work. Our study could contribute to enrich the research of molecular mechanism of NSCLC development, and would provide new targets for NSCLC therapies and potential prognostic factors.

非小细胞肺癌（NSCLC）发生、发展的分子机制是目前研究的热点。研究表明lncRNA在肿瘤的发展过程中起着重要的作用。课题组前期研究发现在NSCLC中lnc-AFAP1-AS1表达显著上调且与临床特征相关；干扰长链后，NSCLC细胞增殖能力明显下降，细胞凋亡增多；课题组过表达c-Myc诱导了长链的上调；文献报道c-Myc可以上调PRC2家族成员EZH2、SUZ12的表达；分别干扰AFAP1-AS1及EZH2、SUZ12，其下游基因表达量明显升高。据此提出假设：原癌基因c-Myc的表达升高，诱导了lnc-AFAP1-AS1及PRC2家族成员的普遍表达上调，两者结合介导了下游一系列抑癌基因的转录沉默，从而导致NSCLC细胞的恶性增殖。本课题组将通过临床样本验证，运用ChIP、RIP、免疫缺陷小鼠等技术证实上述假设，丰富NSCLC发生、发展的分子机制研究，为临床NSCLC诊断、治疗提供理论依据。

非小细胞肺癌（NSCLC）发生、发展的分子机制是目前研究的热点。研究表明lncRNA在肿瘤的发展过程中起着重要的作用。首先，我们在肿瘤术后患者标本中验证AFAP1-AS1表达，结果表明，AFAP1-AS1在癌组织中水平较癌旁显著升高，其差异表达情况与TCGA数据库结果相一致，进一步的分析发现，AFAP1-AS1的表达与肿瘤大小、疾病分期相关，并提示患者的不良预后。随后，通过细胞实验表明，AFAP1-AS1在肺癌细胞株中表达升高，敲低AFAP1-AS1可以抑制细胞增殖，并导致细胞周期阻滞。体内动物实验表明，敲低AFAP1-AS1可以抑制裸鼠种植瘤的成瘤性。我们敲低AFAP1-AS1后进行转录组测序，GO分析发现涉及多个通路的基因表达水平发生了变化，我们发现，敲低AFAP1-AS1后能够诱导p21的表达上调。核质分离实验表明，AFAP1-AS1主要表达于细胞核内，并可以同时绑定PRC2的两个核心蛋白EZH2和SUZ12。敲低AFAP1-AS1或敲低EZH2后，p21的mRNA和蛋白的表达均出现了上调，ChIP实验证实敲低AFAP1-AS1后，可以降低EZH2在p21启动子区的绑定丰度及H3K27三甲基化的水平，且AFAP1-AS1与p21在肿瘤组织中的表达水平呈负相关。我们的研究证实，在NSCLC的发生发展中，lnc-AFAP1-AS1表达升高，通过募集绑定EZH2蛋白，介导了下游包括p21在内的一系列抑癌基因的转录沉默，从而导致NSCLC细胞的恶性增殖。本研究首次阐明例AFAP1-AS1调控的整个转录组基因概况，及其在NSCLC发生发展中的作用机制，基本完成了原定目标。在此青年基金的资助下，本人申请并主持了国家自然科学基金面上项目一项，获市级科技进步二等奖一项，省级新技术引进奖一项，发表SCI论文7篇，其中通讯/共同通讯作者4篇，第一/共同第一作者3篇，在投1篇。在国内学术会议上进行论文投递和学术交流。