一种新的与食管鳞癌预后相关的长链非编码RNA-EPAL的生物学功能及其作用机制的研究

Esophageal squamous cell carcinoma (ESCC) is a prevalent cancer in China and is one of the most deadly cancer forms. Long non-coding RNA (lncRNA) has been reported to have critical roles in cancer formation and development. In a previous study, we examined the lncRNA expression profile in 119 pairs of ESCC and normal tissues and discovered that ample lncRNAs displayed significantly different expression level in ESCC tissues compared with adjacent normal tissues. Further analysis discovered a novel lncRNA significantly down-regulated in ESCC tissues which was associated with lymph node metastasis, TNM stage and prognosis of the patients. We nominate this lncRNA EPAL. Results above showed that EPAL might be involved in the metastasis of the ESCC cells and further affected the survival of ESCC patients. In this study, we plan first to get the whole sequence of EPAL by Northern Blot and RACE. The sub-cellular localization of EPAL will be evaluated by RNA-FISH and RT-PCR. We will then study the effect of EPAL expression on cell proliferation, apoptosis, and invasiveness by over-expressing EPAL in ESCC cell lines. Based on these studies, we will perform RNA-Seq to examine the mRNAs whose expression level might be affected by EPAL. Through the above research, we aim to explore the role of EPAL in the pathogenesis and development of ESCC.

本课题组前期研究发现了在食管鳞癌和癌旁正常组织中异常lncRNA的表达谱，发现了一个尚无功能注释的lncRNA在食管鳞癌组织中表达显著下调，且其表达量与淋巴结转移和患者预后显著相关，命名为EPAL。本研究将首先利用RACE法获取EPAL的cDNA全长序列，并用Northern Blot检测食管鳞癌细胞系和组织以验证RACE结果。进一步通过real time PCR和RNA-FISH明确EPAL的细胞定位。进一步在食管鳞癌细胞中过表达和敲低EPAL表达来研究其对食管鳞癌细胞增殖及转移活性的影响；在此基础上用RNA-Seq分析EPAL的表达改变引起了哪些下游mRNA的转录异常；并在细胞系和组织中验证EPAL的表达异常与下游分子之间的关系，通过改变下游分子表达观察对异常EPAL活性的影响。通过上述研究揭示新的lncRNA EPAL在食管鳞癌发生发展中的作用机制。

非小细胞肺癌（non-small cell lung cancer, NSCLC）发病率和死亡率均位于我国恶性肿瘤第一位。局部浸润和远处转移被认为是NSCLC患者死亡的主要原因。研究表明TGFβ诱导的EMT、免疫耐受及化疗耐药等是NSCLC进展转移的关键通路，但lncRNA在NSCLC TGFβ通路中所扮演的角色仍然不清楚。本研究通过二代高通量全转录组测序比较NSCLC细胞系A549和H226 TGFβ刺激前后长链非编码RNA表达量的变化，并进一步通过RT-qPCR验证及RNAi Screen筛选到促癌的lncRNA TBILA（TGFβ induced lncRNA，RP11757F18.5）进行深入研究。首先我们明确TBILA是位于3号染色体上全长为1698nt定位细胞核的长链非编码RNA，在非小细胞肺癌细胞中受TGFβ/Smad 经典通路的直接调控。体外细胞功能实验发现TBILA能够促进细胞的增殖和侵袭迁移能力，体内小鼠实验证实TBILA能够促进非小细胞肺癌细胞皮下成瘤和远处转移。诱导凋亡实验发现TBILA能够抑制顺铂诱导的细胞凋亡。临床病理研究发现TBILA在非小细胞肺癌组织中显著高表达，且其表达量和生物学功能受到肿瘤微环境中的癌症相关成纤维母细胞（Cancer associated fibroblasts，CAF）分泌的TGFβ所调控。该研究首次揭示了一个全新的 lncRNA TBILA 在非小细胞肺癌中作为癌基因的具体功能和作用机制。 我们的研究结果表明TBILA是TGFβ信号通路的关键调节因子，并揭示TGFβ在调节NSCLC发生发展中lncRNA的重要角色。 对于我们对TGFβ信号通路的促癌机制和NSCLC进展的理解具有重要意义。基于TBILA与非小细胞肺癌临床相关性并在其进展中发挥重要作用，有望成为治疗的有效靶点。