MicroRNA-155/Nrf2对体外培养雪旺细胞增殖和迁移的影响及作用机制

Effects and mechanism of microRNA-155/Nrf2 on proliferation and migration of Schwann cells in vitro

摘要:

目的 探讨microRNA-155（miR-155）/Nrf2对雪旺细胞增殖和迁移的影响及作用机制，为坐骨神经慢性卡压损伤等临床诊断和治疗策略提供科学依据。方法 将大鼠雪旺细胞（RSC96）作为研究对象，转染miR-155 mimics、miR-155 inhibitor、si-Nrf2及其阴性对照质粒（miR-NC、inhibitor NC、si-NC）。为验证miR-155过表达/抑制质粒转染效果，探究miR-155对雪旺细胞增殖、迁移的影响，将细胞分为inhibitor NC组（细胞转染inhibitor NC质粒）、miR-155 inhibitor组（细胞转染miR-155 inhibitor质粒）、miR-NC组（细胞转染miR-NC质粒）、miR-155 mimics组（细胞转染miR-155 mimics质粒）。为了验证Nrf2沉默效果，将细胞分为si-NC组（细胞转染si-NC质粒）、si-Nrf2（1）组［细胞转染si-Nrf2（1）质粒］、si-Nrf2（2）组［细胞转染si-Nrf2（2）质粒］。为证实靶向Nrf2可实现miR-155对细胞增殖、迁移的影响和Nrf2、Ngf、Laminin mRNA相对表达量的调控作用，拟抑制miR-155表达的同时选取Nrf2沉默效果较好的质粒（si-Nrf2）转染细胞行挽救实验，探讨miR-155的作用是否会部分逆转，将细胞分为miR-155 inhibitor+si-NC组（细胞转染miR-155 inhibitor和si-NC质粒）、miR-155 inhibitor+si-Nrf2组（细胞转染miR-155 inhibitor和si-Nrf2质粒）。通过CCK-8法检测miR-155对细胞增殖的影响，Transwell实验检测细胞迁移情况，实时荧光定量聚合酶链反应检测miR-155、神经生长因子（Ngf）、层粘连蛋白（Laminin）和核因子E2相关因子2（Nrf2）的基因水平，Western blotting检测Nrf2蛋白表达，双荧光素酶报告实验验证miR-155与Nrf2的结合关系。结果 miR-155 inhibitor组miR-155相对表达量较inhibitor NC组降低（P <0.05），miR-155 mimics组较miR-NC组升高（P <0.05）。inhibitor NC组、miR-155 inhibitor组、miR-NC组、miR-155 mimics组0 h、24 h、48 h、72 h的吸光度值比较，经重复测量设计的方差分析，结果显示：①不同时间点的细胞增殖有差异（P <0.05）；②各组细胞增殖有差异（P <0.05）；③细胞增殖的变化趋势有差异（P <0.05）。miR-155 inhibitor组细胞迁移数较inhibitor NC组减少（P <0.05），miR-155 mimics组较miR-NC组增加（P <0.05）。miR-155 inhibitor组Ngf、Laminin mRNA相对表达量较inhibitor NC组高（P <0.05），miR-155 mimics组较miR-NC组低（P <0.05）。miR-155 inhibitor组Nrf2 mRNA和蛋白相对表达量较inhibitor NC组升高（P <0.05），miR-155 mimics组较miR-NC组降低（P <0.05）。miR-155 inhibitor野生型Nrf2细胞荧光素酶活性较inhibitor NC组升高（P <0.05），miR-155 mimics组较miR-NC组降低（P <0.05）。si-Nrf2（1）组和si-Nrf2（2）组沉默效果验证实验中Nrf2 mRNA相对表达量较si-NC组降低（P <0.05），且si-Nrf2（2）组效果更好。miR-155 inhibitor组挽救实验中Nrf2 mRNA相对表达量较inhibitor NC组高（P <0.05），miR-155 inhibitor+si-Nrf2组较miR-155 inhibitor+si-NC组低（P <0.05）。miR-155 inhibitor组挽救实验中细胞吸光度值较inhibitor NC组高（P <0.05），miR-155 inhibitor+si-Nrf2组较miR-155 inhibitor+si-NC组低（P <0.05）。miR-155 inhibitor组挽救实验中细胞迁移数量较inhibitor NC组增加（P <0.05），miR-155 inhibitor+si-Nrf2组较miR-155 inhibitor+si-NC组减少（P <0.05）。miR-155 inhibitor组挽救实验中Ngf、Laminin mRNA相对表达量较inhibitor NC组高（P <0.05），miR-155 inhibitor+si-Nrf2组较miR-155 inhibitor+si-NC组低（P <0.05）。结论 miR-155通过调控Nrf2通路活化，抑制雪旺细胞的增殖和迁移。

Abstract:

Objective To explore the effects of microRNA-155 (miR-155)/Nrf2 on proliferation and migration of Schwann cells and its underlying mechanisms, thus providing evidence for clinical diagnosis and treatment of chronic constriction injury of the sciatic nerve.Methods Rat Schwann cells (RSC96) were used for this study and were transfected with miR-155 mimics/inhibitor, si-Nrf2 and their control plasmids (miR-NC, inhibitor NC and si-NC). To testify the efficacy of miR-155 overexpression and inhibition plasmids and the effects of miR-155 on the proliferation and migration of Schwann cells, the cells were divided into inhibitor NC group, miR-155 inhibitor group, miR-NC group and miR-155 mimics group. To determine the efficacy of Nrf2 silencing, the cells were divided into si-NC group, si-Nrf(1) group and si-Nrf(2) group. To confirm the effects of miR-155 on cell proliferation and migration and the regulation of the relative mRNA expressions of Nrf2, Ngf, and Laminin by targeting Nrf2, rescue experiment was performed with si-Nrf2 with better silencing efficacy while inhibiting the expression of miR-155. To investigate whether the effects of miR-155 was reversible, the cells were divided into miR-155 inhibitor + si-NC group and miR-155 inhibitor + si-Nrf2 group. CCK-8 assay was utilized to detect the effect of miR-155 on cell proliferation, while the transwell assay was used to assess cell migration. qRT-PCR was performed to measure the levels of miR-155, Ngf, Laminin and Nrf2. The protein expression of Nrf2 was determined via Western blotting. Dual-luciferase reporter assay was performed to verify the interaction between miR-155 and Nrf2.Results The relative expression of miR-155 in miR-155 inhibitor group was relatively lower than that in inhibitor NC group (P < 0.05), while that in miR-155 mimics group was higher compared with the miR-NC group (P <0.05). The optical density in the inhibitor NC group, miR-155 inhibitor group, miR-NC group and miR-155 mimics group at 0 h, 24 h, 48 h and 72 h was compared via repeated measures ANOVA. The results revealed that cell proliferation was different among the time points (P < 0.05) and between the transfection groups and according negative control groups (P < 0.05), and that the change trends of cell proliferation were also different among the groups (P < 0.05). The cell migration in the miR-155 inhibitor group was decreased compared with the inhibitor NC group (P < 0.05), while that was increased in the miR-155 mimics group compared with the miR-NC group (P < 0.05). The relative mRNA expressions of Ngf and Laminin in the miR-155 inhibitor group were higher than those in the inhibitor NC group (P < 0.05), while they were lower in the miR-155 mimics group than in miR-NC group (P < 0.05). The relative mRNA and protein expression of Nrf2 in the miR-155 inhibitor group were higher than those in the inhibitor NC group (P < 0.05), while they were lower in the miR-155 mimics group than in miR-NC group (P < 0.05). The luciferase activity in the miR-155 inhibitor group was higher than that in the inhibitor NC group (P < 0.05), while that in the miR-155 mimics group was lower than that in the miR-NC group (P < 0.05). The decreases in the relative mRNA expression of Nrf2 in the si-Nrf2(1) group and si-Nrf2(2) group indicated the success in silencing Nrf2 (P < 0.05), and the silencing effect of si-Nrf2(2) was better. In the rescue experiments, the relative mRNA expression Nrf2 in the miR-155 inhibitor was higher than that in the inhibitor NC group (P < 0.05), while that In the miR-155 inhibitor + si-Nrf2 group was lower than that in the miR-155 inhibitor + si-NC group (P < 0.05). The optical density in the miR-155 inhibitor group was higher than that in the inhibitor NC group (P < 0.05), while that in the miR-155 inhibitor + si-Nrf2 group was lower than that in the miR-155 inhibitor + si-NC group (P < 0.05). The cell migration in the miR-155 inhibitor group was increased compared with the inhibitor NC group (P < 0.05), while that in the miR-155 inhibitor + si-Nrf2 group was decreased compared with the miR-155 inhibitor + si-NC group (P < 0.05). In addition, the relative mRNA expressions of Ngf and Laminin in the miR-155 inhibitor group were higher than those in the inhibitor NC group (P < 0.05), while those in the miR-155 inhibitor + si-Nrf2 group were lower than those in the miR-155 inhibitor + si-NC group (P < 0.05).Conclusions miR-155 inhibits the proliferation and migration of Schwann cells by regulating the activation of Nrf2 pathway.