长非编码RNA FEZF1-AS1促进非小细胞肺癌恶性进展的机制研究

By comprehensively analyzing our previous microarray data and the big-data from TCGA datasets, we identified FEZF1-AS1 as a novel long non-coding RNA (lncRNA) associated with human non-small cell lung cancer (NSCLC). We validated the expression level FEZF1-AS1 by real-time RT-PCR and found that it is almost 30-fold up-regulated in NSCLC cancer tissues when comparing with adjacent normal lung tissues. The expression level of FEZF1-AS1 is positively correlated with the TNM stage and lymph node metastasis, and negatively correlated with the long-term survival time of patients significantly. SiRNA-mediated silence of FEZF1-AS1 significantly suppressed the malignant capacity of proliferation, anti-apoptosis, as well as invasion and migration. Furthermore, we identified that FEZF1-AS1 could specifically bind with two RNA binding proteins, HnRNP-K and HnRNP-A2B1 separately, by using RNA pull down and Mass spectrometry screening. RNA immunoprecipitation assay also confirmed the physical interaction between FEZF1-AS1 and two HnRNP proteins. Subsequently, when silencing FEZF1-AS1 by siRNA, CTGF and XIAP, two known target genes of HnRNP-K, were significantly down-regulated. And also β-catenin, a known target gene of HnRNP-A2B1, was down-regulated and which mRNA stability was decreased post-transcriptionally after FEZF1-AS1 silencing. Based on the above preliminary data, we therefore hypothesized that FEZF1-AS1 could promoting NSCLC progression via recruiting HnRNP-K and HnRNP-A2B1 at transcriptional and post-transcriptional level respectively. The current project aims to validate that FEZF1-AS1 could promote NSCLC progression in vitro and in vivo at first. Secondly, the mechanism of FEZF1-AS1 promoting NSCLC progression at both transcriptional and post-transcriptional level will be deeply validated by the experiments of ChIP, RIP, as well as mRNA stability assay. Finally, the correlation between FEZF1-AS1, HnRNP proteins and NSCLC progression will be further validated in a large number of samples of clinical NSCLC patients. To date, no data is available about the function and mechanisms of FEZF1-AS1 promoting NSCLC progression. Our study therefore is the original source of innovation and can provide new potential bio-markers for the prevention and treatment of NSCLC.

FEZF1-AS1（简称FE）是我们综合高通量芯片及TCGA大数据筛选获得的人非小细胞肺癌（简称肺癌）相关长非编码RNA。前期验证显示FE表达在肺癌上调近30倍，与分期及转移正相关，表达越高预后越差；沉默FE可抑制肺癌细胞株增殖侵袭等恶性行为；RNA-蛋白结合及质谱等实验提示：FE可绑定HnRNP-K、激活促增殖基因CTGF及抗凋亡基因XIAP转录，亦可绑定HnRNP-A2B1、转录后维持促转移基因β-catenin的mRNA稳定性。故提出FE绑定两种HnRNP蛋白分别在转录、转录后水平共同促进肺癌恶性进展的科学假说。本项目拟体内外证实FE可促进肺癌恶性进展；分别以两种HnRNP蛋白为线索、设计拯救实验，阐明FE在转录、转录后水平共同促进肺癌恶性进展的分子机制；临床评价FE及两种HnRNP蛋白与肺癌恶性进展的相关性。有关FE促进肺癌恶性进展的功能机制未见报道，本研究可为肺癌防治提供新思路。

背景：FEZF1-AS1（简称F1）是我们综合肺腺癌组织测序及TCGA、GEO第三方数据筛选获得的潜在的人肺腺癌促癌长非编码RNA，迄今尚无研究阐释其在肺腺癌中的功能和具体分子机制。.内容：1.利用肺腺癌新鲜组织PCR及组织芯片原位杂交技术验证F1在肺腺癌中表达情况及其与临床预后的关系；2.通过克隆形成、流式细胞实验、Transwell/Matrigel实验、裸鼠荷瘤/尾静脉转移等体内外功能实验研究F1在肺腺癌中的生物学功能；3.结合ChIP、荧光素酶报告基因等实验探索影响F1表达的上游转录调控机制；4.综合沉默F1后RNA-seq，沉默c-Myc并过表达F1的芯片转录谱以及沉默F1后的ATAC-seq，研究F1影响转录调控的机制；5.应用RNA pull-down、RNA Immunoprecipitation实验正反验证，结合转录活性报告基因实验以及拯救实验阐明F1介导c-Myc转录调控的分子机制。 .结果：1.F1在肺腺癌中普遍高表达，且其表达与不良预后正相关；2.F1能够促进肺腺癌增殖迁移侵袭能力；3.c-Myc能够结合F1启动子并转录激活F1；4.F1能够通过激活AP-1转录活性介导c-Myc下游基因表达以及生物学功能；5.F1能够结合DDX5并促进AP-1转录活性，进而调控CSNK2A1、WNT7B等下游基因转录，介导c-Myc促癌功能。.结论：本课题鉴定了一个肺腺癌高表达的促癌lncRNA—FEZF1-AS1，阐明了F1在细胞核中绑定DDX5，激活AP-1调控CSNK2A1、WNT7B等下游基因转录，从而介导c-Myc促进肺腺癌进展的分子机制。本研究具有源头创新性，可为可为未来c-Myc抑制性治疗，从lncRNA角度开发LUAD核酸治疗药物，提供潜在新靶标。