circ_0001790/miR-509-3-5P/GINS4通路在胃癌生长中的作用及其机制

In the field of gastric cancer, studying the molecular mechanisms of carcinogenesis and progression of gastric cancer, and searching for diagnosis, treatment and prognosis judgment indicators are becoming the hot topics. In the early stage, it was found that GINS4 expression was obviously higher in gastric cancer than that in the adjacent normal tissue through TCGA data base. Additionally, PCR results indicated that GINS4 expression was gradually increased from normal gastric mucosa, precancerous lesion to gastric cancer tissue. Moreover, in vitro experiments revealed that the growth of gastric cancer cells was promoted by GINS4. To clarify the regulation mechanism of GINS4, we found that circ_0001790/miR-509-3-5P pathway may regulate GINS4 by employing the circRNA sequencing, miRNA microarray and bioinformatics assays. Based on the previous results, we put forward the assumption that circ_0001790 relieves the inhibited function of miR-509-3-5P on GINS4 via sponging miR-509-3-5P, and then promotes carcinogenesis and progression of gastric cancer. In order to verify this speculation, we will separately regulate their expression to study the effect of circ_0001790, miR-509-3-5P and GINS4 on the growth of gastric cancer. RIP and other assays will be applied to study upstream and downstream regulation mechanism of GINS4. The expression of circ_0001790, miR-509-3-5P and GINS4 and their relations with the pathology and prognosis of gastric cancer will be validated separately in tissue and plasma. The purpose of this study is to elucidate the effect and mechanism of circ_0001790/miR-509-3-5P/GINS4 pathway in the growth of gastric cancer, and to provide new molecular biomarkers for diagnosis, treatment and prognosis of gastric cancer.

研究胃癌发生发展机制、寻找诊治和预后判断靶点是胃癌研究领域的热点。前期通过TCGA数据库发现GINS4在胃癌组织中表达高于癌旁组织；PCR结果示GINS4在正常胃粘膜、癌前病变、胃癌组织中表达逐渐增高；体外实验发现GINS4促进胃癌细胞生长。为阐明GINS4的调控机制，利用circRNA测序、miRNA芯片和生物信息学分析发现circ_0001790/miR-509-3-5P可能调控GINS4。鉴于此，推测circ_0001790通过海绵样作用吸附miR-509-3-5P，解除其对GINS4的抑制，从而促进胃癌进展。为验证此推测，分别调控三者表达，研究其对胃癌生长的影响；RIP等实验研究GINS4的上、下游调控分子；检测组织和血浆中三者表达与病理和预后的关系。旨在阐明circ_0001790/miR-509-3-5P/GINS4通路在胃癌生长中的作用和机制，发现新的胃癌诊治和预后判断靶点。

在真核生物中，GINS复合体亚单位4（GINS4）在启动DNA复制和延长细胞周期G1/S期中发挥关键作用，但其功能和作用机制尚不清楚。本课题组前期研究发现，GINS4在胃癌组织中表达高于癌旁组织，其表达在正常胃粘膜、癌前病变、胃癌组织中表达逐渐增高，且体外实验发现GINS4能够促进胃癌细胞生长。进一步通过circRNA测序、miRNA芯片和生物信息学分析发现，circ_0001790/miR-509-3-5p可能对GINS4发挥调控作用。基于上述研究基础，本课题组通过联合TCGA数据库、Oncomine数据库及qRT-PCR、Western blotting方法检测GINS4的表达情况，进一步证实GINS4在胃癌组织和细胞中的高表达。结合病理分期等指标发现GINS4表达与肿瘤分化程度、T分期、N分期、疾病分期均呈正相关。K-M生存分析发现高GINS4患者总生存期（Overall survival,OS）和无病生存期（Disease-free survival, DFS）均低于低GINS4胃癌患者。通过上下调细胞中GINS4的表达发现其与生长增殖、细胞周期、细胞凋亡相关。进一步探究发现，GINS4能够直接结合和激活Rac1和CDC42，而circMLLT10（circ_0001790）能够通过结合miR-509-3-5p减弱其对靶标GINS4的抑制作用。同时，circMLLT10促进细胞生长与转移，抑制细胞凋亡，而miR-509-3-5p抑制细胞生长和转移，促进细胞凋亡。这一发现有望为胃癌的精准诊断和治疗提供新的潜在靶点。项目研究成果在Theranostics、Gastric Cancer等期刊发表论文5篇。