miR-219a-5p通过调控NEK6基因表达抑制视网膜母细胞瘤增殖、迁移和侵袭的机制

Mechanism of miR-219a-5p inhibiting proliferation,migration and invasion of retinoblastoma by regulating NEK6 gene expression

目的探讨miR-219a-5p对视网膜母细胞瘤(RB)增殖、迁移和侵袭的影响及作用机制。方法qRT-PCR检测人RB细胞Y79与正常人视网膜血管内皮细胞ACBRI-181中miR-219a-5p和NEK6 mRNA的表达,Western印迹检测NEK6蛋白表达。分别转染miR-219a-5p mimics和si-NEK6至Y79细胞,四甲基偶氮唑蓝(MTT)检测细胞增殖,Transwell检测细胞迁移和侵袭,Western印迹检测周期蛋白依赖性激酶(CDK)1、细胞周期蛋白(Cyclin)D1、基质金属蛋白酶(MMP)-2和MMP-9蛋白表达。双荧光素酶报告基因实验验证miR-219a-5p和NEK6的靶向关系。结果Y79细胞中miR-219a-5p低表达,NEK6 mRNA和蛋白高表达。miR-219a-5p过表达或抑制NEK6表达可抑制Y79细胞增殖、迁移和侵袭,下调Y79细胞CDK1、Cyclin D1、MMP-2和MMP-9蛋白表达。miR-219a-5p在Y79细胞中靶向负调控NEK6表达,NEK6过表达可逆转miR-219a-5p过表达对Y79细胞增殖、迁移和侵袭的抑制作用。结论miR-219a-5p调控NEK6表达抑制Y79细胞的增殖、迁移和侵袭能力,是治疗RB的新靶点。

Objective To investigate the effect and mechanism of miR-219a-5p on proliferation,migration and invasion of retinoblastoma(RB).Methods The expressions of miR-219a-5p and NEK6 mRNA in human RB cell Y79 and normal human retinal vascular endothelial cells ACBRI-181 were detected by qRT-PCR.The expression of NEK6 protein was detected by Western blot.The miR-219a-5p mimics and si-NEK6 were transfected into Y79 cells respectively.MTT was used to detect cell proliferation,Transwell was used to detect cell migration and invasion,and Western blot was used to detect CDK1,Cyclin D1,MMP-2 and MMP-9 protein expression.The dual luciferase reporter gene assay validated the targeting relationship between miR-219a-5p and NEK6.Results The level of miR-219a-5p was low,and the levels of NEK6 mRNA and protein were high in Y79 cells.Over-expression miR-219a-5p or inhibition of NEK6 expression in Y79 cells inhibited the proliferation,migration and invasion of Y79 cells,and down-regulated the expression of CDK1,Cyclin D1,MMP-2 and MMP-9 proteins.miR-219a-5p negatively regulated NEK6 expression in Y79 cells,and over-expression of NEK6 reversed the inhibitory effect of miR-219a-5p over-expression on proliferation,migration and invasion of Y79 cells.Conclusions miR-219a-5p could inhibit the proliferation,migration and invasion of Y79 cells by regulating NEK6 expression,and which is a new target for the treatment of RB.