长非编码RNA SBF2-AS1调控食管鳞癌增殖的机制研究

Through the analysis of TCGA database our research group identified a novel lncRNA SBF2-AS1 (SBF), which is highly over-expressed in esophageal squamous cell carcinoma(ESCC) and other solid tumors. Our previous study found that the expression level of SBF is positively correlated with tumor size and tumor stage, and negatively correlated with the long-term survival time of patients; SiRNA-mediated silence of SBF significantly suppressed the capacity of proliferation, also significantly arrested cell cycle at the G1 stage; Bioinformatics analysis and previous experiments indicated that SBF located in cytoplasm and could upregulate cell cycle related key gene E2F1 expression by competing binding miR-338-3p/miR-362-3p in post transcriptional level. We therefore hypothesized that SBF could upregulate E2F1 expression to promote ESCC proliferation by acting as sponge of miR-338-3p/miR-362-3p. The current project aims to assess the relationship between SBF expression level and ESCC proliferation by clinical samples. Over-expression and knockdown manners were used to demonstrated that SBF could promote the proliferation of ESCC both in vivo and in vitro. And then, the molecular mechanism network of competing endogenous RNA between SBF, miR-338-3p/miR-362-3p, and E2F1 will be validated by rescue experiment. The function and mechanism of SBF in ESCC has not been reported, our study offers a new clue of prevention and treatment of ESCC.

SBF2-AS1（简称SBF）是课题组结合TCGA大数据筛选获得、在食管鳞癌等多种实体肿瘤显著高表达的长非编码RNA。前期发现：食管鳞癌样本中SBF表达与肿瘤大小及分期显著正相关，且表达越高预后越差；体外沉默SBF能抑制食管鳞癌细胞增殖、诱导G1期阻滞；生物信息学及预实验证实SBF定位胞浆、可通过吸附miR-338-3p及362-3p转录后上调细胞周期关键基因E2F1表达。故提出SBF通过竞争性内源RNA（ceRNA）机制吸附miR-338-3p及362-3p上调E2F1表达促进食管鳞癌增殖的科学假说。本项目拟结合临床样本评价SBF与食管鳞癌增殖的相关性；采取过表达/沉默策略，结合细胞实验及动物模型证实SBF可促进食管鳞癌增殖；设计拯救实验，阐明SBF竞争性吸附两种miRNA上调E2F1表达形成ceRNA网络的分子机制。有关SBF在食管鳞癌中的功能机制未见报道，本研究可为其防治提供新线索。

LncRNA是当前肿瘤研究领域的热点，国内外不断有关于lncRNA参与调控肿瘤发生发展的重磅论文发表。食管癌是一种恶性程度很高的肿瘤，目前食管鳞癌的治疗方法虽然多种多样，但总体预后仍很差，因此研究lncRNA对食管鳞癌调控的分子机制具有重大意义。本课题研究发现lncRNA SBF2-AS1通过ceRNA网络机制参与了对食管鳞癌增殖的调控。通过TCGA数据库寻找到了在多种肿瘤中发挥促癌作用的长链非编码RNA-SBF2-AS1，并结合生物信息学和分子生物学等方法和技术，对SBF2-AS1调控食管鳞癌增殖的分子机制进行研究。首先通过RT-PCR发现SBF2-AS1在食管鳞癌组织和细胞系中高表达并发现其高表达与食管鳞癌患者不良预后相关，可作为判断食管鳞癌患者不良预后的独立因素。通过一系列的功能实验验证SBF2-AS1可以促进食管鳞癌增殖，之后通过FISH实验发现SBF2-AS1主要定位于胞浆，这是SBF2-AS1作为ceRNA发挥作用的前提条件之一。紧接着通过生物信息学分析找到了SBF2-AS1两个下游靶基因（miR-338-3P和miR-362-3P），并利用RT-PCR实验证实miR-338-3P和miR-362-3P在食管鳞癌细胞系中低表达。并通过RIP和pulldown等实验证实SBF2-AS1可以作为ceRNA发挥作用，并和miR-338-3P/miR-362-3P之间存在互补序列。众所周知lncRNA、miR-RNA和mRNA是构成ceRNA网络的核心，因此利用生物信息学分析查找是否存在miR--338-3P和miR-362-3P的靶RNA，结果发现E2F1可以作为miR-338-3P/miR-362-3P的下游靶基因。利用双荧光素酶报告基因实验和pulldown双向验证了miR-338-3P和miR-362-3P与E2F1之间的结合。一系列的拯救实验Western blot实验验证了SBF2-AS1是通过竞争性结合miR-338-3P和miR-362-3P来调控E2F1的表达从而促进食管鳞癌增殖。综合上述结果，本研究为SBF2-AS1作为食管鳞癌治疗及判断预后提供了理论依据。