p53通过长链非编码RNA LOC401317和CREB5蛋白抑制舌鳞癌细胞增殖的机制研究

Recent studies have revealed that long non-coding RNAs (lncRNAs) are involved in the TP53 regulatory network. Using tongue squamous cell carcinoma (TSCC) as a research model, we transfected the TP53 gene into TSCC cells and analyzed the expression of all known coding and non-coding genes using gene chips. We found that expression of the lncRNA LOC401317 was most significantly altered in these samples and that its expression was very similar to that of the protein-coding gene CREB5, which is located only 1767bp downstream of LOC401317. We predicted the promoter of LOC401317 using bioinformatic tools, and found that there is one potential promoter in the upstream sequence, and there are TP53 and CREB5 transcription factor binding sites in the promoter region, implying that LOC401317 may be directly redulated by TP53 and CREB5. Using clinical samples, we verified that TP53 mutations are correlated with low expression of LOC401317 and CREB5, all of which predict poor prognostic outcomes for TSCC. Preliminary experiments showed that LOC401317 could inhibit the proliferation of TSCC cells by regulating CREB5. In the proposed project, we will examine the regulatory mechanisms among TP53, LOC401317 and CREB5, and we will determine how LOC401317 inhibits the proliferation of TSCC cells by regulating CREB5. Achieveing the goals in this proposal will further elucidate our understanding of the TP53 regulatory network as well as provide new prognostic markers and therapeutic targets for TSCC treatment.

近年来的研究发现lncRNAs参与了抑瘤基因TP53的调控网络。我们以舌鳞癌为模型，在舌癌细胞中转染TP53并检测所有编码和非编码基因的表达，发现lncRNA LOC401317表达变化最显著，且LOC401317的表达与其下游仅1767bp的CREB5基因紧密相关。通过生物信息学预测，发现LOC401317的潜在启动子区域存在TP53和CREB5的结合位点，推测LOC401317受TP53和CREB5直接调控。我们在临床样本中证实TP53突变与LOC401317及CREB5的低表达相关且都是舌鳞癌的不良预后因素，初步证明LOC401317可通过CREB5抑制舌鳞癌细胞增殖。本项目拟研究TP53、LOC401317和CREB5之间的调控机制，及LOC401317通过CREB5抑制舌鳞癌细胞增殖的分子机制。本项目的完成将进一步丰富TP53基因调控网络，并提供新的舌鳞癌预后分子标记和治疗靶点。

本课题在舌鳞癌细胞Tca8113中过表达TP53基因，通过lncRNA芯片检测，筛选出了TP53基因调控的差异表达的lncRNAs分子，其中LOC401317显著上调。生物信息学分析及荧光素酶报告基因活性检测证实，p53 通过与LOC401317转录起始位点上游p53结合位点结合，直接调控LOC401317的表达。在舌鳞癌细胞中过表达LOC401317，发现LOC401317能明显抑制肿瘤细胞的增殖；且 LOC401317通过促进细胞G1/S期阻滞和诱导细胞凋亡，抑制肿瘤细胞的增殖；裸鼠移植成瘤实验在体内证实，LOC401317能抑制肿瘤细胞的生长。进一步的研究发现，LOC401317可能通过作用于p21 和cyclins阻滞细胞周期进程，通过依赖caspase的机制诱导细胞凋亡，从而发挥肿瘤抑制作用。在口腔癌组织标本中检测lncRNA LOC401317的表达水平，发现LOC401317在肿瘤组织中表达下调，且低表达LOC401317的肿瘤患者比高表达LOC401317的患者预后差。本课题还利用舌鳞癌细胞中过表达TP53基因后lncRNAs和miRNAs表达谱芯片检测结果，进行生物信息学分析并在舌鳞癌和鼻咽癌细胞中检测相关分子的表达水平，构建了TP53基因调控的miRNAs、mRNAs和lncRNAs相互作用网络。通过本课题的研究，已初步阐明TP53 基因通过lncRNA LOC401317 抑制舌鳞癌细胞增殖的分子机制，相关结果已在SCI期刊 (PLoS ONE、Tumour Biol等) 上发表。由于LOC401317具有重要的抑瘤作用，其可望成为舌鳞癌基因治疗潜在的新靶点，为舌鳞癌的治疗提供一条新的途径。

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