川楝素通过调节circ_0032627/miR-502-5p轴对胃癌侵袭转移的作用及其分子机制

Tumor metastasis is the leading cause of the death of patients with gastric cancer. Therefore, it is one of key issues to explore the molecular mechanisms and therapeutic strategies of gastric cancer metastasis. Our previous studies showed that, the active ingredient toosendanin extracted from the root bark of toosendan exerts antitumor effects on gastrointestinal tumors. Increasing data indicate that circular RNAs (circRNAs), a subgroup of noncoding RNA with covalently closed loop structures are involved in the regulation of tumor progression. Herein, circRNA expression profiling and bioinformatics were conducted for analysis of toosendanin-regulated circ_0032627/miR-502-5p axis in GC cells, and silencing of circ_0032627 suppressed tumor proliferation and colony formation. Based on circ_0032627 targeted miR-502-5p regulation pathway, our project intends to investigate the molecular mechanisms underlying gastric cancer metastasis and the intervention of toosendanin by using bioinformatics, fluorescence in situ hybridization, RNA Pull-down assay, RIP, luciferase assay as well as establishing gastric cancer with liver metastasis, caudal vein pulmonary metastasis and subcutaneous xenograft tumor models. These findings may provide the experimental and theoretical basis for improving the prognosis of patients with gastric cancer.

肿瘤转移是导致胃癌患者死亡的主要原因，研究其侵袭转移的调控机制及治疗策略是当前亟待解决的关键问题之一。我们前期研究发现，中药川楝子有效成分川楝素具有抗胃肠道肿瘤作用。近年研究表明，环状RNA (circular RNAs, circRNAs)作为一种共价闭合的非编码RNA参与肿瘤的发生发展。我们前期利用circRNAs表达谱和生物信息学鉴别胃癌细胞中川楝素调控的circ_0032627/miR-502-5p轴，且沉默circ_0032627抑制胃癌增殖和克隆集落形成。故本课题拟利用生物信息学、荧光原位杂交技术、RNA Pull-down、RIP、luciferase等手段，构建胃癌肝转移、尾静脉肺转移及皮下移植瘤等模型，基于circ_0032627靶向miR-502-5p调控径路，探讨川楝素影响胃癌侵袭转移的作用及其分子机制，为改善胃癌患者预后提供实验及理论依据。

肿瘤转移是导致胃癌患者死亡的主要原因，研究其侵袭转移的调控机制及治疗策略是当前亟待解决的关键问题之一。我们前期研究发现，中药川楝子有效成分川楝素具有抗胃肠道肿瘤作用。近年研究表明，环状RNA (circular RNAs, circRNAs)作为一种共价闭合的非编码RNA参与肿瘤的发生发展。我们应用荧光原位杂交（FISH）分析胃癌患者中hsa_circ_0032627 (circDLST)的表达水平和细胞定位及其与临床病理特征和预后的关系。细胞体外MTT，克隆形成实验，Transwell及EdU等实验以及皮下移植肿瘤模型和SCID原位胃癌肝转移模型评估circDLST在胃癌细胞中的作用。通过双荧光素酶基因报告、RNA免疫沉淀（RIP）分析和RIP-miRNA表达谱，证实CircDLST与miR-502-5p的特异性结合。qRT-PCR和Western blot分析用于检测circDLST对胃癌细胞中miR-502-5p介导的NRAS/MEK1/ERK1/2信号传导的影响。定量PCR和细胞功能实验检测川楝素介导circDLST抗胃癌作用。结果：与邻近正常组织相比，circDLST在胃癌组织中的表达水平显著升高，并作为胃癌患者生存不良的独立预后因素。circDLST的敲除在体外和体内抑制了细胞活力、集落形成、DNA合成、细胞侵袭和肝转移，而circDLST的过表达则逆转这些效果。此外，circDLST与miR-502-5p在胃癌细胞的细胞质中共定位，并在胃癌细胞中充当miR-502-3p的海绵，后者可使胃癌细胞中的NRAS/MEK1/ERK1/2信号失活并消除circDLST的促肿瘤作用。另外，川楝素可通过下调circDLST发挥抗胃癌作用。结论：CircDLST通过吸附miR-502-5p和激活NRAS/MEK1/ERK1/2信号促进胃癌发生和转移，而川楝素可通过下调circDLST发挥抗胃癌作用。我们的研究为改善胃癌患者预后提供实验及理论依据。