摘要
目的分析胃癌细胞释放外泌体在缺氧诱导胃癌进展中的作用和分子调控机制。方法通过超速离心法提取胃癌细胞培养基中外泌体,利用电子显微镜、免疫印迹法对外泌体进行鉴定,RT-PCR分析外泌体、胃癌细胞中miR-143-5p水平。构建miR-143-5p antagomir/angomir分别抑制、增强胃癌细胞中miR-143-5p的表达。双萤光素酶报告基因验证miR-143-5p与下游SMDT1 mRNA的偶联。利用细胞划痕愈合实验分析胃癌细胞迁移、侵袭能力变化,细胞克隆形成实验分析胃癌细胞增殖能力变化。结果缺氧诱导胃癌细胞HGC、AGS高分泌外泌体促进了常氧胃癌细胞的增殖克隆、迁移和侵袭能力增加。缺氧诱导胃癌细胞及外泌体高表达miR-143-5p,并且通过外泌体转导并促使常氧胃癌细胞的miR-143-5p上调。在线数据库Starbase中胃癌组织miR-143-5p呈明显高表达趋势,而且与胃癌患者不良预后明显正相关。萤光素酶报告基因预测并证实了miR-143-5p与抑癌基因SMDT1 mRNA的偶联结合,并且竞争性抑制SMDT1 mRNA转录表达。富含miR-143-5p的缺氧缓解外泌体被常氧胃癌细胞内吞,并通过抑制SMDT1表达,可间接激活PI3K/Akt途径促进了这些常氧胃癌细胞的增殖、侵袭和迁移。相反,通过转染miR-143-5p antagomir抑制miR-143-5p表达,或使用PI3K抑制剂LY294002阻断PI3K/Akt通路可有效抑制缺氧胃癌细胞衍生的外泌体介导的促癌作用。结论胃癌中的缺氧微环境可能促进肿瘤细胞释放富含miR-143-5p的外泌体,进而被常氧胃癌细胞以内吞方式吸收并最终促进癌细胞的侵袭、克隆增殖。
Objective To explore the roles and molecular mechanism of hypoxia induced exosome release and the promoting progression of gastric cancer.Methods Exosomes of gastric cancer cells pre-treated with hypoxia were extracted, and identified by electron microscopy and Western blotting. The levels of miR-143-5 p in exosomes and cancer cells were determined by RT-PCR, while miR-143-5 p antagomir and miR-143-5 p angomir were transfected to inhibiting and enhancing the miR-143-5 p, respectively. Then, the dual luciferase reporter was applied to verify the coupling of miR-143-5 p and SMDT1 mRNA. The cell migration and invasion abilities were evaluated by scratch healing experiment. Cell colony formation was used to determine the cell proliferation ability. Results The exosomes derived from hypoxic gastric cancer HGC and AGS cells contained high level of miR-143-5 p, and promoted the proliferation, migration and invasion of normoxic gastric cancer cells. Data of Starbase predicted that miR-143-5 p showed a significant high expression in gastric cancer tissues, and high level of miR-143-5 p was positively correlated with poor prognosis of patients with gastric cancer. The hypoxic exosomes containing high level of miR-143-5 p were endocytosed by normoxic AGS, HGC cells and activated the PI3 K/Akt pathway by suppression the expression of SMDT1 mRNA, thereby, promoting the proliferation, invasion and migration of these normoxic gastric cancer cells. On the contrary, inhibition of miR-143-5 p by miR-143-5 p antagomir or blocking the PI3 K/Akt signaling by LY294002 could effectively reduce the tumor progression mediated by exosomes derived from hypoxic gastric cancer cells.Conclusion The hypoxic microenvironment in gastric cancer maypromote the release of miR-143-5 p-rich exosomes from tumor cells. These exosomes transferred to normoxic gastric cancer cells to enhance tumor progression.
作者
刘伟
胡永波
白少华
穆磊
李珊
LIU Wei;HU Yongbo;BAI Shaohua;MU Lei;LI Shan(Department of Gastrointestinal Surgery,Xiantao First People’s Hospital Affiliated to Yangtzeu University,Xiantao 433000;Department of Gastrointestinal Surgery,Tongji Hospital,Tongji Medical College,Huazhong University of Science and Technology;Department of Endocrinology,Xiantao First People’s Hospital Affiliated to Yangtzeu University,China)
出处
《胃肠病学和肝病学杂志》
CAS
2021年第12期1331-1338,共8页
Chinese Journal of Gastroenterology and Hepatology
基金
湖北省青年人才项目(WJ2019-09)。
作者简介
第一作者:刘伟,主治医师,研究方向:胃肠外科常见肿瘤研究。E-mail:hbxtlw2020@126.com;通讯作者:李珊,主治医师,研究方向:肿瘤相关内分泌机制。E-mail:hbxtlishan2020@163.com。
