长非编码RNA LINC00346作为胃癌生物标志物及促进胃癌进展的分子机制研究

Long non-coding RNA is closed related to the progression of gastric cancer (GC). We found that LINC00346 expression was significantly upregulated in GC tissues than in the adjacent normal tissues by analyzing Gene Expression Omnibus (GEO) database and it has been verified in collected tissues. We further found that LINC00346 expression was also remarkably elevated in exosomal RNA derived from serum of GC patients than that of health subjects by next generation sequencing technology. Knockdown of LINC00346 could significantly inhibit proliferation and metastasis of GC cell, and increase E-cadherin expression, decrease CDK4, CDK7, CD44, MET or ZEB1 expression. Cell cycle analysis revealed that inhibition of LINC00346 expression had an obvious cell-cycle arrest in GC cells. We found that LINC00346 promoter regions contained KLF5, MYC, JUN/FOS, and SP1 binding sites by analyzing GEO and ENCODE database. Bioinformatics was shown that LINC00346 could bind to miR-34a, miR-181c, miR-143, miR-340 and miR-205 with lower binding energy, and the above miRNAs were upregulted upon LINC00346 depletion. RNA immunoprecipitation assay was confirmed that LINC00346 binded to Argonaute2, the core subunits of RNA-induced silencing complex (RISC). CDK4, CDK7, CD44, MET or ZEB1 are the target genes of above miRNAs. Accordingly, the hypothesis is proposed: Oncogenic transcription factors promotes LINC00346 expression, and LINC00346 regulates CDK4, CDK7, CD44, MET or ZEB1 expression by competing for related miRNAs to promote the progression of GC.We will document this hypothesis by bioinformatic analysis and laboratory testing. Moreover, our work will further the understanding about the molecular mechanism of GC progression and provides a new basis of early diagnosis and molecular targeted therapy for GC.

长非编码RNA与胃癌进展相关。对在线数据库分析发现LINC00346在胃癌（GC）组织中高表达，测序技术发现LINC00346在GC患者血清外泌体中表达较对照者上调。敲降LINC00346抑制胃癌细胞增殖及转移，下调CDK4、CD44、MET及ZEB1等表达。在线数据库分析发现转录因子（KLF5、MYC、JUN/FOS及SP1）绑定LINC00346启动子区。生物信息学发现LINC00346与miRNAs结合，敲低LINC00346上调相关miRNAs，RIP证实LINC00346与miRNA效应蛋白Ago2结合，CDK4、CD44、MET及ZEB1等是相应miRNAs靶基因。据此提出假设：促癌因子促进LINC00346表达，其通过竞争吸附相关miRNAs调控下游靶基因，促进胃癌进展。本课题组将利用生物信息学预测及实验验证相结合的方法证实上述假设，为GC早期诊断及分子靶向治疗提供依据。

课题组通过分析多基因表达综合数据库中的胃癌数据，发现了胃癌中异常表达的长非编码RNA 346（LINC00346）。LINC00346在胃癌中异常扩增和上调，其表达与不良病理分期、肿瘤体积增大和预后不良呈正相关。此外，致癌转录因子KLF5和MYC可以与LINC00346启动子区结合并增强其表达。基因集富集分析显示细胞周期和粘着斑相关基因在LINC00346高表达的患者中富集。通过体外和体内实验，课题组发现LINC00346会影响包括细胞生长，迁移和侵袭在内的复杂表型。高通量测序分析发现，LINC00346改变会引起胃癌细胞中细胞周期和粘着斑通路的异常。LINC00346可以募集argonaute 2蛋白，吸附miR-34a-5p并抑制其功能，从而阻遏CD44，NOTCH1和AXL的翻译。总而言之，我们的研究发现了影响肿瘤发生和进展的关键生物标志物，即KLF5，MYC / LINC00346 / miR-34a-5p，为胃癌治疗提供了新的研究方向。