MicroRNA-9通过NRP1抑制胃癌SGC-7901细胞上皮-间充质转化功能

MicroRNA-9 inhibits epithelial-mesenchymal transition in gastric cancer SGC-7901 cells by NRP1

目的:研究微小RNA-9(microRNA-9,miR-9)对胃癌SGC-7901细胞上皮-间充质转化(EMT)功能的影响及其相关机制。方法:SGC-7901胃癌细胞株分别转染miR-9 mimics和阴性对照序列(negative control mimic,NCM),作为miR-9组和NCM组,并设立未转染对照(control)组,采用RT-qPCR法检测各组细胞miR-9的含量,Transwell实验检测3组细胞迁移能力和侵袭能力,Western blot法检测3组细胞的N-cadherin、E-cadherin、α-catenin和神经纤毛蛋白1(NRP1)表达水平。采用Western blot法检测NRP1过表达对miR-9抑制EMT的拮抗作用。双萤光素酶实验检测miR-9与NRP1的关系。结果:miR-9组的miR-9表达水平明显上调,为control组的538倍(P<0.05)。miR-9组的迁移细胞数量明显低于control组(P<0.05)。miR-9组的侵袭细胞数量明显低于control组(P<0.05)。miR-9组细胞的N-cadherin和NRP1蛋白表达量明显降低,E-cadherin及α-catenin蛋白表达量明显升高。而NRP1及miR-9均过表达组胃癌细胞中N-cadherin蛋白表达量明显升高,E-cadherin及α-catenin蛋白表达量明显降低。双萤光素酶检验结果显示NRP1为miR-9的下游靶基因(P<0.05)。结论:miR-9可能通过降低下游靶基因NRP1水平影响EMT相关蛋白表达,抑制胃癌SGC-7901细胞的EMT功能。

AIM: To investigate the inhibitory effect of microRNA-9(miR-9) on epithelial-mesenchymal transition(EMT) in the gastric cancer SGC-7901 cells and its mechanism. METHODS: The gastric cancer cell line SGC-7901 was transfected with miR-9 mimics or negative control mimic(NCM),as miR-9 or NCM group,respectively. The SGC-7901 cells without transfection were used as control group. The expression level of miR-9 in each group was detected by RT-qPCR. The migration and invasion abilities of the SGC-7901 cells in the 3 groups were detected by Transwell assay.The protein expression of N-cadherin,E-cadherin,α-catenin and neuropilin-1(NRP1) was determined by Western blot.Antagonistic effect of NRP1 over-expression on miR-9 inhibition of EMT was detected by Western blot. The relationship between miR-9 and NRP1 was analyzed by dual luciferase assay. RESULTS: The expression level of miR-9 in miR-9 group was significantly up-regulated,which was 538 times higher than that in control group(P<0.05). The number of migratory cells in miR-9 group was significantly lower than that in control group( P<0.05). Compared with control group,the protein expression of N-cadherin and NRP1 in miR-9 group was significantly decreased,while the protein expression of E-cadherin and α-catenin protein was significantly increased. Over-expression of NRP1 resulted in the increase in the protein expression of N-cadherin in the gastric cancer cells of miR-9 group,and the decrease in the protein expression of E-cadherin and α-catenin significantly. The result of dual luciferase assay showed that NRP1 was a downstream target gene of miR-9(P<0.05). CONCLUSION: miR-9 may inhibit the expression of EMT-related proteins through the downstream target gene NRP1,thus inhibiting the EMT of gastric cancer SGC-7901 cells.