tRNA衍生片段tRF-24-V29K9UV3IU及其介导的调控网络在胃癌侵袭和转移中的作用机制研究

Invasion and metastasis of gastric cancer is the main cause leading to death. However, its mechanism has not been fully elucidated. tRNA derived fragments (tRFs) are a newly discovered non-coding RNA that plays an important role in tumor invasion and metastasis. However, there is no tRFs reported in gastric cancer till now. In our previous study, we found that tRF-24 was significantly downregulated in gastric cancer tissue compared with that in adjacent tissue by small RNA sequencing and real-time PCR. In gastric cancer cell lines, overexpression of tRF-24 inhibits the metastasis and invasion of gastric cancer cells and promotes apoptosis. Based on these results, we speculate that the low expression of tRF-24 might be closely related to invasion and metastasis of gastric cancer. In this study, we aimed to detect the expression of downstream target genes and proteins and the proliferation, migration and apoptosis of gastric cancer cells by overexpression of tRF-24 in gastric cancer cells. Besides, the effect of tRF-24 overexpression on tumor growth was assessed by constructing xenograft model. The transcriptome sequencing was further used to investigate the target gene regulatory network after tRF-24 overexpression and to verify the function of tRF-24 by various molecular biological experiments. This study can provide prognostic markers for the prevention and treatment of gastric cancer, which has a good innovation and scientific significance.

胃癌的侵袭和转移是其致死的主要原因，但相关机制尚未完全阐明。tRNA衍生片段（tRNA derived fragments,tRFs）是一种新发现的在肿瘤侵袭与转移中起重要作用的非编码RNA，迄今在胃癌中还没有相关tRFs的报道。我们利用小RNA测序和定量PCR发现tRF-24-V29K9UV3IU在胃癌组织中表达量显著降低。在胃癌细胞系中，过表达tRF-24会抑制胃癌细胞转移和侵袭，促进细胞凋亡。由此，我们推测tRF-24低表达可能与胃癌侵袭转移密切相关。本项目拟通过在胃癌细胞中过表达tRF-24，检测下游靶基因、蛋白的表达及胃癌细胞增殖迁移凋亡等功能变化，并通过构建小鼠异位瘤模型，检测过表达tRF-24对移植瘤生长的影响；通过转录组测序研究tRF-24靶基因调控网络并通过多种分子生物学实验验证其生物学功能。本研究可为胃癌防治提供诊断预后标志物及治疗新靶点，具有较好的创新性及科学意义。

项目背景：tRNA衍生片段（tRNA derived fragments,tRFs）是一种新发现的在肿瘤侵袭与转移中起重要作用的非编码RNA，迄今在胃癌中还没有相关tRF-24-V29K9UV3IU的报道。.主要研究内容：①采用Small RNA测序对胃癌组织和癌旁组织进行测序，筛选鉴定出与胃癌进展密切相关的tRFs。②在临床样本中检测tRF-24-V29K9UV3IU表达，确定其临床意义。③过表达及沉默tRF-24-V29K9UV3IU，利用CCK-8 检测胃癌细胞增殖、transwell检测细胞迁移及侵袭、流式细胞仪检测细胞凋亡，探讨tRF-24-V29K9UV3IU对胃癌细胞生物学行为的影响。④在动物模型中检测 tRF-24-V29K9UV3IU的体内功能。⑤利用转录组测序结合生信预测，确认tRF-24-V29K9UV3IU 调控的靶基因。荧光素酶报告基因、AGO-RIP、PCR、WB验证 tRF-24-V29K9UV3IU与靶基因相互作用关系。⑥通过拯救实验检测tRF-24-V29K9UV3IU靶向调控靶基因对胃癌细胞生物学功能的影响，明确tRF-24-V29K9UV3IU抑制胃癌侵袭转移的分子机制。.重要结果及关键数据：①本研究通过高通量测序筛选鉴定出与胃癌进展密切相关的 tRF-24-V29K9UV3IU。②tRF-24-V29K9UV3IU 的表达水平在胃癌组织中显著下调，对于胃癌和非癌组织具有一定的鉴别与诊断价值。③过表达tRF-24-V29K9UV3IU 显著抑制胃癌细胞的增殖、迁移和侵袭，增强凋亡。④沉默tRF-24-V29K9UV3IU 显著增强胃癌细胞的增殖、迁移和侵袭，抑制凋亡水平。⑤沉默 tRF-24-V29K9UV3IU 促进体内胃癌的成瘤和转移。⑥tRF-24-V29K9UV3IU 以类似 miRNA 的机制，通过靶向调控 GPR78 抑制胃癌细胞增殖、迁移和侵袭，促进细胞凋亡。.科学意义及应用前景：我们的研究揭示胃癌组织和非癌组织间的 tRFs 表达谱存在差异，tRF-24-V29K9UV3IU对于胃癌和非癌组织具有一定的鉴别与诊断价值，有望成为胃癌的诊断标志物。tRF-24-V29K9UV3IU 通过 GPR78 发挥抑癌作用的机制，可能成为治疗胃癌的一个新的潜在靶点。