miR-340对神经母细胞瘤细胞增殖、迁移的影响及其作用机制研究

Effects of miR-340 on proliferation and migration of neuroblastoma cells and its mechanism

目的探讨微小RNA-340(miR-340)对神经母细胞瘤细胞(SH-SY5Y)增殖、迁移的影响及其作用机制。方法选择2018年10月至2020年12月在延安大学附属医院进行手术治疗的23例神经母细胞瘤及其瘤旁组织标本进行实验研究;体外培养SH-SY5Y细胞,将细胞分为对照组、miR-340过表达(miR-340 mimics)组、miR-340阴性对照组(negative control,NC)组、miR-340 mimics+pcDNA组及miR-340 mimics+SKP2组,分别检测肿瘤组织和瘤旁组织、各组SH-SY5Y细胞miR-340、SKP2mRNA表达及各组细胞增殖、迁移侵袭及SKP2、基质金属蛋白酶2(MMP-2)、MMP-9表达;双荧光素酶报告实验验证miR-340与SKP2的靶向关系。结果神经母细胞瘤组织中miR-340表达水平为0.55±0.08,明显低于瘤旁组织的1.05±0.15,SKP2 mRNA表达水平为1.96±0.20,明显高于瘤旁组织的1.01±0.11,差异均有统计学意义(P<0.05);双荧光素酶报告实验表明miR-340与SKP2存在靶向调控关系;与对照组和miR-340 NC组相比,miR-340 mimics组SH-SY5Y细胞miR-340表达水平显著升高,细胞存活率、划痕迁移率、侵袭数、SKP2 mRNA和蛋白表达、MMP-2、MMP-9蛋白表达水平显著降低,差异均有统计学意义(P<0.05);与miR-340 mimics组和miR-340 mimics+pcDNA组比较,miR-340 mimics+SKP2组细胞存活率、划痕迁移率、侵袭数、SKP2 mRNA及蛋白、MMP-2、MMP-9蛋白表达水平明显升高,差异均有统计学意义(P<0.05)。结论神经母细胞瘤中miR-340低表达,过表达miR-340可抑制SH-SY5Y细胞增殖、迁移与侵袭,其机制可能与下调SKP2表达有关。

Objective To investigate the effect of microRNA-340(miR-340)on the proliferation and migration of neuroblastoma cells(SH-SY5Y)and its mechanism.Methods Neuroblastoma and adjacent tissue specimens were collected from 23 patients who underwent surgical treatment in the Affiliated Hospital of Yan'an University from October 2018 to December 2020.SH-SY5Y cells were cultured in vitro,and the cells were divided into control group,miR-340 overexpression(miR-340 mimics)group,miR-340 negative control(negative control,NC)group,miR-340 mimics+pcDNA group,and miR-340 mimics+SKP2 group.The expression of miR-340 and SKP2 mRNA,cell proliferation,migration and invasion,and the expression of SKP2,matrix metalloproteinase 2(MMP-2),and MMP-9 in SH-SY5Y cells of each group and tumor tissues and adjacent tissues were detected.Dual-luciferase reporter assay was conducted to verify the targeting relationship between miR-340 and SKP2.Results The expression level of miR-340 in neuroblastoma tissue was 0.55±0.08,which was significantly lower than 1.05±0.15 in adjacent tissue;the expression level of SKP2 mRNA was 1.96±0.20,which was significantly higher than 1.01±0.11 in the adjacent tissue;the differences were statistically significant(P<0.05).Dual-luciferase reporter assay showed that miR-340 has a targeted regulatory relationship with SKP2.Compared with the control group and the miR-340 NC group,the expression level of miR-340 in SH-SY5Y cells in the miR-340 mimics group was significantly increased,while cell survival rate,scratch migration rate,invasion number,SKP2 mRNA and protein expression,MMP-2,MMP-9 protein expression levels were significantly decreased,with statistically significant difference(P<0.05).Compared with miR-340 mimics group and miR-340 mimics+pcDNA group,cell survival rate,scratch migration rate,invasion number,SKP2 mRNA and protein,MMP-2,MMP-9 protein expression levels of miR-340 mimics+SKP2 group significantly increased(P<0.05).Conclusion The expression of miR-340 is low in neuroblastoma.Overexpression of miR-340 can inhibit the proliferation,migration,and invasion of SH-SY5Y cells,and the mechanism may be related to down-regulation of SKP2 expression.