低甲基化上调长非编码RNA FOXD2-AS1调控NOTCH信号通路促进胃癌进展的分子机制

Tumor recurrence and metastasis mainly account for gastric cancer (GC)-related deaths.Accumulating data has been reported that long non-coding RNA (lncRNA) is closed related to progression of GC. We found that FOXD2-AS1 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. Knock down of FOXD2-AS1 could significantly inhibit proliferation and metastasis of GC cell, and increase E-cadherin expression, decrease Notch1，Twist or p-STAT3 expression. Cell cycle analysis revealed that inhibition of FOXD2-AS1 expression had an obvious cell-cycle arrest in GC cells. Computational analysis predicted a CpG island spanning the transcription initiation site in FOXD2-AS1 gene, FOXD2-AS1 was upregulated after demethylation treatment of 5-Aza. The methylation status of the FOXD2-AS1 gene was assayed in GC cells. The normal gastric cell line reveals a methylated FOXD2-AS1 promoter, whereas its methylation status in GC cell lines is decreased. RNA immunoprecipitation assay was confirmed that FOXD2-AS1 binded Argonaute2, the core subunits of RNA-induced silencing complex (RISC). Bioinformatics was shown that FOXD2-AS1 could bind miR-34a and miR-139 with lower binding energy, while Notch1 is the target gene of above miRNAs. Accordingly, the hypothesis is proposed: LncRNA FOXD2-AS1 hypomethylation results in an increase of its expression, and FOXD2-AS1 regulates Notch1 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.

对GEO数据库分析发现FOXD2-AS1在胃癌组织中表达较癌旁组织显著上调，并在收集的组织标本中得到验证。敲降FOXD2-AS1抑制胃癌细胞增殖及转移，并下调Notch1。生物信息学发现FOXD2-AS1启动子区含有CpG岛，MS-PCR发现胃癌细胞系FOXD2-AS1启动子区甲基化程度较正常胃粘膜细胞系低，去甲基化药物处理上调FOXD2-AS1。生物信息学发现FOXD2-AS1与miR-34a及miR-139结合，敲低FOXD2-AS1上调上述miRNAs，RIP证实FOXD2-AS1与Ago2结合，并且Notch1是上述miRNAs靶基因。据此提出假设：FOXD2-AS1基因启动子区低甲基化促使其在胃癌中高表达，其通过竞争性吸附miRNAs而上调靶基因Notch1的表达，促进胃癌进展。课题组将利用生物信息学预测及实验室验证相结合的方法证实上述假设，为胃癌早期诊断及分子靶向治疗提供依据。

大量的数据表明长非编码RNA（lncRNA）在生物过程中起重要的调节作用，并且在不同的肿瘤中表达失调。FOXD2-AS1在胃癌进展中的功能和相关的生物学机制仍未明确。综合分析发现，FOXD2-AS1在胃癌中显著上调，并与肿瘤大小、病理分期偏晚和预后不良呈正相关。GEO数据集中的基因集富集分析（GSEA）揭示了在高FOXD2-AS1表达的患者中细胞周期和DNA复制相关基因富集明显。敲低FOXD2-AS1可通过抑制胃癌细胞周期进程来抑制细胞生长，而上调FOXD2-AS1表达可促进癌症进展。我们发现zeste同源物2（EZH2）和赖氨酸（K）特异性脱甲基酶1A（LSD1）蛋白充当了FOXD2-AS1的结合配体并介导FOXD2-AS1的功能。在机制上，FOXD2-AS1部分通过EZH2和LSD1介导的EphB3下调促进胃癌发生。目前的研究结果表明：FOXD2-AS1通过与EZH2和LSD1的直接相互作用，部分通过抑制EphB3而促进胃癌进展，并且可能作为肿瘤发生的潜在生物标志物。