lncRNA ROR1-AS1通过靶向miR-758-3p调控神经母细胞瘤SK-N-SH细胞的增殖、迁移、侵袭和凋亡

lncRNA ROR1-AS1 Regulates the Proliferation,Migration,Invasion and Apoptosis of Neuroblastoma Cells SK-N-SH by Targeting miR-758-3p

该研究旨在探讨ROR1-AS1对神经母细胞瘤细胞SK-N-SH增殖、迁移、侵袭和凋亡的影响及作用机制。收集67例神经母细胞瘤组织和瘤旁组织,实时荧光定量PCR(RT-qPCR)检测组织中ROR1-AS1和miR-758-3p的表达情况。转染ROR1-AS1小干扰RNA、miR-758-3p模拟物或共转染ROR1-AS1小干扰RNA与miR-758-3p抑制剂至SK-N-SH细胞,细胞计数试剂盒-8(CCK-8)法检测细胞增殖,Transwell检测细胞迁移和侵袭,流式细胞术检测细胞凋亡,蛋白质印迹法检测CyclinD1、p21、Bcl-2、Bax、MMP-2和MMP-9的蛋白表达情况。双荧光素酶报告基因实验验证ROR1-AS1和miR-758-3p的调控关系。结果显示,神经母细胞瘤组织中ROR1-AS1的表达明显高于瘤旁组织,而miR-758-3p表达明显低于瘤旁组织。抑制ROR1-AS1表达或过表达miR-758-3p降低了SK-N-SH细胞活性、迁移数和侵袭数及CyclinD1、Bcl-2、MMP-2和MMP-9蛋白表达水平,而提高了细胞凋亡率及p21和Bax蛋白表达水平。ROR1-AS1在SK-N-SH细胞中靶向负调控miR-758-3p表达,干扰miR-758-3p可逆转抑制ROR1-AS1对SK-N-SH细胞增殖、迁移、侵袭和凋亡的影响。这提示抑制ROR1-AS1表达可能通过靶向上调miR-758-3p阻碍SK-N-SH细胞增殖、迁移和侵袭,并促进细胞凋亡,ROR1-AS1有可能成为神经母细胞瘤治疗的分子靶点。

The purpose of this study was to explore the effect and mechanism of ROR1-AS1 on the proliferation,migration,invasion and apoptosis of neuroblastoma cells SK-N-SH.Sixty-seven cases of neuroblastoma tissue and adjacent tissues were collected,and RT-qPCR was used to detect the expression levels of ROR1-AS1 and miR-758-3p in the tissues.ROR1-AS1 small interfering RNA or miR-758-3p mimics were transfected into SK-N-SH cells,or ROR1-AS1 small interfering RNA and miR-758-3p inhibitor were co-transfected into SK-N-SH cells.CCK-8 method was used to detect cell proliferation.Transwell was used to detect cell migration and invasion.Flow cytometry was used to detect cell apoptosis,and Western blot was used to detect the protein expression levels of CyclinD1,p21,Bcl-2,Bax,MMP-2 and MMP-9.The dual luciferase reporter gene experiment verified the regulatory relationship between ROR1-AS1 and miR-758-3p.The results showed that the expression of ROR1-AS1 in neuroblastoma tissues was higher than that in adjacent tissues,but the expression of miR-758-3p was lower than that in adjacent tissues.Inhibiting ROR1-AS1 or overexpressing miR-758-3p reduced the activity of SK-N-SH cells,the number of migrating cells,the number of invasion cells,as well as the protein expression of CyclinD1,Bcl-2,MMP-2 and MMP-9 in cells.But inhibiting ROR1-AS1 or overexpressing miR-758-3p increased the apoptosis rate of SK-N-SH cells and the protein expression of p21 and Bax in cells.ROR1-AS1 negatively regulated the expression of miR-758-3p in SK-N-SH cells.Interfering with miR-758-3p reversed the effect of inhibiting ROR1-AS1 on proliferation,migration,invasion and apoptosis of SK-N-SH cells.This suggested that inhibiting ROR1-AS1 might block the proliferation,migration and invasion of SK-N-SH cells and promote cell apoptosis by targeting to up-regulate the expression of miR-758-3p.ROR1-AS1 may become a molecular target for neuroblastoma treatment.