Exosome内非编码RNAs检测在胃癌诊疗中的应用及机制

Exosomal non-coding RNA (miRNA and lncRNA), as emerging non-invasive markers for the diagnosis and therapy of cancers, have attracted increasing attention. Previously, we found that the levels of serum exosomal non-coding RNA (miR-423-5p and 7SKRNA) derived from GC-MSC exosome were significantly increased in patients with gastric cancer compared to that in healthy control. Our results also showed that miR-423-5p could promote EMT and metastasis of gastric cancer cells. However，the clinical significance of exosomal non-coding RNAs in diagnosis and treatment of gastric cancer remains to be evaluated. Therefore, in this proposal we aim to establish a new method for detecting exosomal non-coding RNAs , then we will assess the value of separate and combined detection of exosomal non-coding RNAs in diagnosis of gastric cancer compared with traditional methods. The roles and mechanisms of exosomal non-coding RNA will be explored by using cell and animal models as well as CRISPR/Cas9 technique. Our study will provide new methods and novel biomarkers for gastric cancer diagnosis, reveal the roles and mechanisms of these exosomal non-coding RNAs in cancer metastasis, and provide new therapeutic targets.

Exosome内非编码RNA（miRNA和lncRNA）作为一种新的非创伤性分子标志物在肿瘤诊疗中的作用备受关注。我们检测到胃癌微环境间质干细胞分泌的exosome内miR-423-5p及7SKRNA等非编码RNAs在胃癌患者血清exosome内显著增高，并发现miR-423-5p可促进胃癌细胞转移及上皮细胞间质化，但其在胃癌诊疗中的应用价值还有待评估。本研究拟采用qRT-PCR方法检测胃癌患者血清exosome内miR-423-5p及7SKRNA等非编码RNAs的表达水平，建立分子实验室诊断新技术，与传统方法比较并进行方法学评价，评估其单独和联合检测在胃癌诊疗中的应用价值；采用基因转染及CRISPR/Cas9基因敲除技术，结合细胞和动物模型解析exosome内miR-423-5p及7SKRNA在胃癌转移中的作用及机制。将为胃癌诊疗提供新技术和分子标志，为胃癌诊治提供新靶标，具有重要意义。

Exosome内非编码RNAs和蛋白质在肿瘤的诊断、疗效观察及预后判断中具有潜在的临床应用价值。本项目前期检测到胃癌微环境间质干细胞分泌的exosome内miR-423-5p及RMRP在胃癌患者血清exosome内显著增高。在此基础上，本研究扩大样本量进行验证，成功提取胃癌患者及健康体检者血清来源exosome、胃癌细胞及胃粘膜上皮细胞来源exosome。血清及细胞来源的exosome为圆形或椭圆形小囊泡，直径介于40~150nm，表面表达CD9、CD63分子。胃癌患者血清exosome内miR-423-5p的表达水平较健康体检者显著升高。胃癌患者及健康体检者联合胃癌细胞及胃粘膜上皮细胞共同验证血清及细胞来源的exosome内miR-423-5p表达具有显著差异。ROC曲线分析其诊断效能高于胃癌传统肿瘤标志物CEA、CA-199。结合胃癌患者临床病理资料分析发现，胃癌患者血清exosome内miR-423-5p的表达水平与淋巴结转移显著相关，而与性别、年龄、肿瘤直径大小、分化程度和TNM分期无关。Exosome能够传递miR-423-5p至胃癌细胞发挥促胃癌增殖及转移的作用。生物信息学软件预测结合实验验证确定miR-423-5p候选靶基因为SUFU，miR-423-5p通过与SUFU的mRNA3’-UTR位点结合靶向负调控SUFU。胃癌血清exosome内RMRP表达明显高于健康体检者血清exosome，并且在胃癌细胞来源exosome内RMRP的表达也较胃粘膜上皮细胞增高。Exosome能够介导RMRP的传递，促进胃癌的增殖和迁移能力。项目拓展了exosome内其他分子标志物的研究如TRIM3等。本研究为临床exosome常规检测研究提供实验室新方法，为胃癌诊断提供新型非创伤性分子标志物，为胃癌的治疗提供新的靶点，具有重要的科学意义和临床应用前景。.本项目累计发表14篇SCI论文（6篇影响因子大于5,2篇影响因子大于10）和1篇中文核心期刊；获得科技奖励4项；参加国内外学术交流7次；培养博士研究生3名，硕士研究生4名。项目投入经费60.0000万元，支出52.0821万元，各项支出基本与预算相符。