MiR-107靶向Notch2促进胃癌细胞迁移和侵袭

MicroRNA-107 promotes migration and invasion of gastric cancer cells by targeting Notch2

目的检测微小RNA-107(miR-107)在胃癌细胞中的表达,探讨其调控胃癌细胞迁移和侵袭的分子机制。方法采用Real-time PCR检测miR-107和Notch2在人胃癌细胞MKN45和人胃黏膜上皮细胞GES-1中的表达。转染miR-107 inhibitors至MKN45细胞,采用Transwell法检测MKN45细胞迁移和侵袭。转染Notch2过表达载体至MKN45细胞,采用Transwell、Real-time PCR、Weatern blot分别检测细胞迁移、侵袭和基质金属蛋白酶-9(matrix metalloproteinase-9,MMP-9)的表达。Targetscan在线预测和双荧光素酶报告基因实验验证miR-107和Notch2的靶向关系。Transwell实验检测抑制miR-107并沉默Notch2后MKN45细胞迁移和侵袭。结果与GES-1细胞比较,MKN45细胞中miR-107表达上调(P<0.01),而Notch2表达下调(P<0.01)。抑制miR-107或过表达Notch2,则MKN45细胞迁移和侵袭数减少(P<0.01),过表达Notch2能促进MMP-9 mRNA和蛋白的表达(P<0.01)。Targetscan在线预测和双荧光素酶报告基因实验结果表明:Notch2是miR-107的潜在靶基因,抑制miR-107可促进Notch2表达(P<0.01)。在MKN45细胞中沉默Notch2,可逆转miR-107抑制剂对细胞迁移和侵袭的抑制作用。结论 miR-107在胃癌细胞中表达上调,可通过靶向Notch2促进MMP-9表达,促进胃癌细胞迁移和侵袭。

Objective To detect the expression of microRNA-107(miR-107) in gastric cancer cells and its molecular mechanism for regulating migration and invasion of gastric cancer cells. Methods The expression levels of miR-107 and Notch2 mRNA in gastric cancer MKN45 cells and gastric epithelial cells GES-1 were detected using real-time PCR. MKN45 cells were transfected with a miR-107 inhibitor and the changes in cell migration and invasion were detected with Transwell assay. In MKN45 cells transfected with a Notch2-overexpressing plasmid, the changes in cell migration and invasion and the expression of matrix metalloproteinase-9(MMP-9) were detected using Transwell assay, real-time PCR and Western blotting, respectively. Targetscan online prediction and dual-luciferase assay were used to analyze the relationship between miR-107 and Notch2. The changes in the migration and invasion of MKN45 cells after inhibiting miR-107 and Notch2 silencing were investigated using Transwell assay. Results Compared with those in GES-1 cells, the expression of miR-107 was significantly up-regulated(P<0.01) and Notch2 was significantly down-regulated(P<0.01) in MKN45 cells. Both inhibition of miR-107 and overexpression of Notch2 significantly attenuated the migration and invasion abilities of MKN45 cells(P<0.01). Overexpression of Notch2 significantly promoted the expressions of MMP-9 mRNA and protein in the cells(P<0.01). The results of Targetscan online prediction and dual-luciferase assay showed that Notch2 was potentially the target gene of miR-107(P<0.01), and inhibiting miR-107 promoted the expression of Notch2. Silencing Notch2 in MKN45 cells obviously reversed miR-107-induced inhibition of cell migration and invasion. Conclusion The expression of miR-107 is up-regulated in gastric cancer cells. MiR-107 enhances the expression of MMP-9 by targeting Notch-2 to promote the migration and invasion of gastric cancer cells.