Yan Chao, Zhang Lian-Hai, Shan Fei, Li Shuang-Xi, Jia Yong-Ning, Li Zi-Yu
Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Gastrointestinal Cancer Center, Peking University Cancer Hospital and Institute, Beijing 100142, China.
Zhongguo Yi Xue Ke Xue Yuan Xue Bao. 2018 Aug 30;40(4):528-533. doi: 10.3881/j.issn.1000-503X.10687.
Objective To detect the expression of microRNA(miR)-199 in gastric carcinoma tissues and cell lines, and further explore the effect and molecular mechanism of miR-199 on the proliferation and migration of gastric carcinoma cell lines. Methods Reverse transcriptase-polymerse chain reaction was used to detect the expression of miR-199 in gastric carcinoma and adjacent normal tissue obtained from 51 patients and in gastric carcinoma cell lines and human gastric epithelial cell line GES-1. The gastric carcinoma cell lines over-expressing and low-expressing miR-199 were established to detect their proliferation and migration abilities. Dual-luciferase reporter assay was performed to detect the regulatory effect of miR-199 on the 3'untranslated region of TBL1XR1. Western blot was used to explore the miR-199-related mechanism. Results The relative expression of miR-199 in gastric carcinoma tissues was significantly lower than that in the adjacent normal tissue (0.2635±0.0303 vs. 1.6700±0.9613, t=13.95, P<0.001). The relative expressions of miR-199 in gastric carcinoma cell lines AGS (0.81, t=9.13, P<0.001), SGC-7901 (0.83, t=8.88, P<0.001), MKN28 (0.58, t=10.80, P<0.001), KATO-3 (0.60, t=10.31, P<0.001), MKN-45 (0.27, t=13.10, P<0.001) were significantly lower than that in the normal gastric cell line GES-1 (2.1). In miR-199 over-expressed cell lines, the cell proliferation and migration significantly decreased as compared with the control group of gastric carcinoma cells (731±13 vs. 345±18, t=24.90, P<0.001), and in miR-199 low-expressed group, the cell proliferation and migration increased compared with the control group of gastric carcinoma cells (257±16 vs. 657±8, t=32.59, P<0.001). Dual-luciferase reporter assay proved that miR-199 directly targeted on the 3' untranslated region of TBL1XR1. Western blot analysis showed that miR-199 inhibited the expression of TBL1XR1. Conclusion The over-expression of miR-199 in gastric carcinoma is associated with the decreased ability of proliferation and migration of gastric carcinoma cells by targeting TBL1XR1.
目的 检测微小RNA（miR）-199在胃癌组织及细胞系中的表达，并进一步探讨miR-199对胃癌细胞系增殖和迁移的影响及其分子机制。方法 采用逆转录-聚合酶链反应检测51例患者胃癌及癌旁正常组织、胃癌细胞系及人胃上皮细胞系GES-1中miR-199的表达。构建过表达和低表达miR-199的胃癌细胞系，检测其增殖和迁移能力。采用双荧光素酶报告基因检测法检测miR-199对TBL1XR1 3′非翻译区的调控作用。运用蛋白质免疫印迹法探讨miR-199相关机制。结果 miR-199在胃癌组织中的相对表达量显著低于癌旁正常组织（0.2635±0.0303 vs. 1.6700±0.9613，t=13.95，P<0.001）。miR-199在胃癌细胞系AGS（0.81，t=9.13，P<0.001）、SGC-7901（0.83，t=8.88，P<0.001）、MKN28（0.58，t=10.80，P<0.001）、KATO-3（0.60，t=10.31，P<0.001）、MKN-45（0.27，t=13.10，P<0.001）中的相对表达量均显著低于正常胃细胞系GES-1（2.1）。在miR-199过表达细胞系中，与胃癌细胞对照组相比，细胞增殖和迁移能力显著降低（731±13 vs. 345±18，t=24.90，P<0.001）；在miR-199低表达组中，与胃癌细胞对照组相比，细胞增殖和迁移能力增强（257±16 vs. 657±8，t=32.59，P<0.001）。双荧光素酶报告基因检测法证实miR-199直接靶向TBL1XR1的3′非翻译区。蛋白质免疫印迹分析显示miR-199抑制TBL1XR1的表达。结论 胃癌中miR-199的过表达通过靶向TBL1XR1与胃癌细胞增殖和迁移能力降低相关。
