miR-216b-5p靶向调控BTN3A2促进胶质瘤细胞LN-229的迁移及侵袭能力

miR-216b-5p Promotes the Migration and Invasion of LN-229 Glioma Cells by Targeting BTN3A2

大量证据表明microRNA(miRNA)通过靶向调控靶基因的表达从而在肿瘤侵袭与转移中发挥重要作用。然而关于microRNA-216b-5p (miR-216b-5p)通过靶向嗜乳脂蛋白第3亚家族膜蛋白A2(butyrophilin subfamily 3 member A2,BTN3A2)促进胶质瘤侵袭与转移的机制尚不明确。本研究通过GSE15824与GSE4290差异表达分析筛选出同时在2个芯片中表达上调的BTN3A2(P<0.05)。生存曲线结果显示,高表达BTN3A2病人总生存期明显下降(P<0.001)。表达量分析结果显示,BTN3A2表达随WHO分级升高而升高(P<0.05),同时1p/19q未联合缺失与IDH突变型病人BTN3A2表达升高(P<0.001)。基因集富集分析(gene set enrichment analysis,GSEA)结果显示,BTN3A2与众多癌症相关通路有关(P<0.05);Western印迹结果显示,BTN3A2在7例胶质瘤组织和胶质瘤细胞系U87、U251和LN-229中表达上调,过表达miR-216b-5p(miR-216b-5p mimics)后BTN3A2蛋白表达水平降低;Transwell结果显示,转染BTN3A2干扰质粒(si-BTN3A2)和miR-216b-5p mimics后可以抑制LN-229细胞体外迁移与侵袭能力(P<0.05);在线预测网站证实,miR-216b-5p为BTN3A2潜在靶基因;生存曲线结果显示,与低表达miR-216b-5p病人相比,高表达病人生存率明显上调(P=0.025);荧光定量RT-PCR结果显示,miR-216b-5p在胶质瘤U87、U251和LN-229细胞中表达下降(P<0.05);双荧光素酶结果显示,BTN3A2存在与miR-216b-5p的结合靶点(P<0.05);综上所述,BTN3A2可能通过结合miR-216b-5p促进胶质瘤细胞LN-229的迁移以及侵袭。

Accumulating evidence indicated that microRNAs(miRNA) play an important role in tumor invasion and metastasis by regulating their target genes. However, whether the miRNA-216 b-5 p(miR-216 b-5 p) and their target genes butyrophilin subfamily 3 member A2(BTN3 A2) promote glioma invasion and metastasis is unclear. This study aims to study whether miR-216 b-5 p promoted migration and invasion in glioma cells by negatively regulating BTN3 A2. The differential expression analysis of GSE15824 and GSE4290 was analyzed by GEO2 R. We found that only BTN3 A2 is up-regulated in both GSE15824 and GSE4290(P<0.05). The gene set enrichment analysis(GSEA) analysis indicated BTN3 A2 was related to many cancer-related pathways(P<0.05). The results of survival curves showed that the overall survival of patients with high expression of BTN3 A2 decreased significantly(P <0.001). The expression of BTN3 A2 was increased with the increase of WHO grade(P<0.05), while the expression of BTN3 A2 was increased in 1 p/19 q uncombined deletion and IDH mutant patients(P<0.001). Western blotting results showed that BTN3 A2 was up-regulated in seven glioma tissues and glioma cell lines U87, U251 and LN-229 and down-regulated in the miR-216 b-5 p mimics group;Transwell results showed that transfection with BTN3 A2 silencing plasmids(si-BTN3 A2) or miR-216 b-5 p mimics plasmids could inhibit the ability of migration and invasion in LN-229 cells in vitro(P<0.05). The online websites predicted miR-216 b-5 p as a potential target gene of BTN3 A2. The survival curve results show that compared with patients with low expression of miR-216 b-5 p, the survival rate of patients with high expression was significantly increased(P=0.025). The relative expression of miR-216 b-5 p was decreased in U87, U251 and LN-229 cells was detected by real time quantitative PCR(P<0.05). The results of dual luciferase assay showed that BTN3 A2 could bind to miR-216 b-5 p(P<0.05). Transwell experiment results showed that overexpression of miR-216 b-5 p can inhibit the migration and invasion ability of LN-229 cells(P<0.05). In summary, miR-216 b-5 p promotes the migration and invasion by targeting BTN3 A2 of LN-229 glioma cells.