长非编码RNA FAM83H-AS1调控非小细胞肺癌恶性进展的机制研究

Combining the analysis of our previous microarray data with the large RNA-seq data mining from TCGA datasets, we identified FAM83H-AS1 as a novel long non-coding RNA (lncRNA) which is highly over-expressed in human non-small cell lung cancer (NSCLC) tissues. Further expression level validation of this lncRNA was performed by real-time RT-PCR and we found that FAM83H-AS1 is almost 20-fold upregulated in lung cancer tissues when comparing with adjacent normal lung tissues. The expression level of FAM83H-AS1 is positively correlated the tumor size and lymph node metastasis, and negatively correlated with the long-term survival time of patients significantly. SiRNA-mediated silence of FAM83H-AS1 significantly suppresses the malignant capacity of proliferation, anti-apoptosis, invasion, and migration and also significantly induces that cell cycle arrest at the G1/S transition. Furthermore, both bioinformatics, RNA-seq data and our preliminary studies suggested that the expression of FAM83H-AS1 is positively correlated with the expression of a well-known oncogene, c-Myc. We further found that FAm83H-AS1 could reserve the mRNA stability of c-Myc via recruiting a RNA binding protein, hnRNP-A1. Moreover, c-Myc is also able to activate the transcription of FAM83H-AS1. We therefore hypothesized there might be a reciprocal regulation between FAM83H-AS1 and c-Myc which forming a feed-forward loop and finally promoting NSCLC progression. The current project aims to validate that FAM83H-AS1could promote NSCLC progression in vitro and in vivo by using silencing and overexpressing strategy at first. Secondly, the mechanism of a feedforward loop between FAM83H-AS1and c-Myc will be deeply validated by the experiments of ChIP, RIP, as well as mRNA stability assay. Finally, the correlation between FAM83H-AS1/c-Myc loop 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 FAM83H-AS1 promoting NSCLC progression, therefore, our study is the original source of innovation and can provide new potential biomarkers for the prevention and treatment of NSCLC.

前期应用芯片及TCGA大数据分析筛选获得一条差异高表达于人非小细胞肺癌（简称肺癌）组织、功能未知的长非编码RNA：FAM83H-AS1（简称FA）。验证显示FA显著高表达于肺癌组织，与肿瘤大小及转移正相关，且表达越高预后越差；沉默FA可抑制肺癌细胞株增殖侵袭等恶性行为；软件分析及预实验提示：与肿瘤恶性进展密切相关的明星分子c-Myc和FA表达高度正相关，FA可能通过招募hnRNP-A1维持c-Myc mRNA稳定性，c-Myc亦可激活FA转录。故提出FA与c-Myc形成正反馈调控环路促进肺癌恶性进展的科学假说。本项目拟采用过表达/沉默策略，体内外证实FA可促进肺癌恶性进展；结合ChIP、RIP、mRNA稳定性等实验，阐明FA与c-Myc形成正反馈调控环路的分子机制；临床评价FA/c-Myc环路与肺癌恶性进展的相关性。有关FA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。

近年来，生命科学研究已跨入后基因组时代，约占基因组转录产物70-90%的长非编码RNA（lncRNA），能在表观遗传、转录及转录后等多个水平发挥调控作用，因而受到极大关注。恶性肿瘤是目前lncRNA 研究最为活跃的领域之一，因此，从lncRNA 角度继续挖掘NSCLC 新型靶标并深入探讨分子机制十分必要。. 对于在非小细胞肺癌组织及细胞系中高表达的长链非编码RNA FAM83H-AS1，课题组使用泛肿瘤基因组学的方法，对FAM83H-AS1的生物学特性进行了深入的挖掘。通过对癌症基因组图谱（TCGA）肺腺癌数据基因组及转录组进行联合分析，并结合机器学习等方法，发现基因组8q24区域存在促癌lncRNA簇，结果表明FAM83H-AS1是一个潜在的肺腺癌驱动基因。通过RACE实验，我们首次测定了FAM83H-AS1在肺腺癌中的转录本全长，结果区别于目前已知的参考序列。而后续的机制实验表明，FAM83H-AS1可结合RNA结合蛋白HNRNPK，而高表达的FAM83H-AS1会促进HNRNPK进入细胞质发挥生物学功能。我们对敲降FAM83H-AS1的肺腺癌细胞系进行了高通量表达谱与蛋白谱的分析，结果提示是蛋白水平而非转录水平的变化与表型具有高相关。结合高通量数据并查阅相关文献，我们发现FAM83H-AS1很可能通过促进癌基因RAB8B与RAB14的蛋白翻译来发挥生物学功能。而随后的实验证实了FAM83H-AS1/HNRNPK/RAB8B&RAB14的分子机制作用轴。课题组最后建立了小鼠移植瘤模型（PDX），并以FAM83H-AS1为靶点进行了转化研究，结果表明，敲降FAM83H-AS1后，荷瘤体积明显缩小，肿瘤细胞凋亡显著增加。. 本研究对肺腺癌高表达lncRNA的表达水平、基因组扩增水平及临床预后的关系进行了深入的分析，鉴定出FAM83H-AS1等多个潜在的肺腺癌驱动lncRNA。并首次对FAM83H-AS1的分子作用机制进行了深入的探索，证实其通过结合RNA结合蛋白HNRNPK促进下游靶基因的蛋白表达，并显著抑制肺腺癌细胞的凋亡。本研究具有源头创新性，从lncRNA的角度为开发肺腺癌核酸治疗药物提供潜在的新靶标。