LncRNA UNC5B-AS1对胶质母细胞瘤细胞增殖、迁移、侵袭的影响及与microRNA-199的关系

Effect of lncRNA UNC5B-AS1 on proliferation, migration and invasion of glioblastoma cell lines and its relationship with microRNA-199

摘要:

目的 探究长链非编码RNA UNC5B-AS1（LncRNA UNC5B-AS1）对胶质母细胞瘤（GBM）细胞增殖、迁移、侵袭的影响及与microRNA-199（miR-199）的关系。方法 体外培养GBM细胞系U251，分为对照组、空载组、抑制组及过表达组。对照组细胞不做处理；空载组、抑制组、过表达组分别采用空载质粒、si-LncRNA UNC5B-AS1、过表达LncRNA UNC5B-AS1载体转染GBM细胞系U251。qRT-PCR检测LncRNA UNC5B-AS1、miR-199的表达；CCK-8法、划痕实验、Transwell实验分别检测U251细胞增殖、迁移及侵袭能力；双萤光素酶法检测LncRNA UNC5B-AS1与miR-199的靶向作用；Western blotting检测PI3K/Akt通路蛋白的表达。结果 与对照组、空载组比较，抑制组LncRNA UNC5B-AS1降低（P <0.05），miR-199升高（P <0.05），过表达组LncRNA UNC5B-AS1升高（P <0.05），miR-199降低（P <0.05）。与对照组、空载组比较，抑制组细胞活力指数、划痕愈合率、细胞侵袭数、p-PI3K/PI3K、p-Akt/Akt蛋白相对表达量降低（P <0.05），过表达组细胞活力指数、划痕愈合率、细胞侵袭数、p-PI3K/PI3K、p-Akt/Akt蛋白相对表达量升高（P <0.05）。双萤光素酶实验结果显示，LncRNA UNC5B-AS1与miR-199-mimics基因上存在结合靶点。结论 LncRNA UNC5B-AS1在GBM细胞中高表达，抑制LncRNA UNC5B-AS1能够抑制GBM细胞的增殖、迁移、侵袭作用，其作用机制可能与靶向调控miR-199和调节PI3K/Akt通路有关。

Abstract:

Objective To investigate the effects of LncRNA UNC5B-AS1 on the proliferation, migration and invasion of glioblastoma (GBM) cell lines and its relationship with microRNA-199 (miR-199).Methods GBM cell line U251 was cultured in vitro and divided into control group, blank group, inhibition group and overexpression group. The control group was left untreated, the blank group was transfected with empty plasmid vectors, the inhibition group was transfected with si-LncRNA UNC5B-AS1, and the overexpression group was transfected with LncRNA UNC5B-AS1 overexpression vectors. The expression levels of LncRNA UNC5B-AS1 and miR-199 were detected by quantitative real-time polymerase chain reaction. The proliferation, migration and invasion ability of U251 cells were detected by CCK8 assay, scratch assay and transwell assay, respectively. The interaction between LncRNA UNC5B-AS1 and miR-199 was determined via dual-luciferase assays. The expressions of proteins associated with the PI3K/Akt pathway were detected via Western blotting.Results Compared with the control group and the blank group, the expression of LncRNA UNC5B-AS1 was lower and the expression of miR-199 was higher in the inhibition group (P < 0.05), while the expression of LncRNA UNC5B-AS1 was higher and the expression of miR-199 was lower in the overexpression group (P < 0.05). Compared with the control group and the blank group, the cell viability index, the number of invasive cells, the rate of scratch wound healing, and the relative protein expressions of p-PI3K/PI3K and p-Akt/Akt were lower in the inhibition group (P < 0.05), whereas the cell viability index, the number of invasive cells, the rate of scratch wound healing, and the relative protein expressions of p-PI3K/PI3K and p-Akt/Akt were higher in the overexpression group (P < 0.05). The dual-luciferase assays showed that there were binding targets on LncRNA UNC5B-AS1 and miR-199 genes for their interactions.Conclusions LncRNA UNC5B-AS1 is highly expressed in GBM cell lines, and inhibition of LncRNA UNC5B-AS1 could suppress the proliferation, migration and invasion of GBM cell lines, which may be achieved via the targeted regulation of miR-199 and the modulation of the PI3K/Akt pathway.