miR-34a-5p靶向BCAN对胶质瘤细胞增殖、迁移的影响

【摘要】目的探究MicroRNA-34a-5p（miR-34a-5p）过表达对胶质瘤细胞增殖、迁移及侵袭的影响及其与短蛋白聚糖（BCAN）的靶向关系。方法收集36例脑胶质瘤患者的脑胶质瘤组织和因脑外伤行内减压术的36例患者的正常脑组织。体外培养人胶质瘤细胞U373、U251 MG、LN229、A172和人正常星形胶质细胞NHA，逆转录定量PCR（RT-qPCR）检测组织/细胞中miR-34a-5p、BCAN mRNA表达水平。取对数生长期的U251 MG细胞，分为对照组（NC组）、mimic-NC组（转染miR-34a-5p mimic阴性对照）、miR-34a-5p过表达组（转染miR-34a-5p mimic）、miR-34a-5p过表达+pcDNA3.1-NC组（miR-34a-5p mimic和pcDNA3.1-BCAN阴性对照空载体质粒共转染）、miR-34a-5p过表达+pcDNA3.1-BCAN组（miR-383-5p mimic和pcDNA3.1-BCAN共转染）。转染48 h后，收集各组细胞用RT-qPCR检测转染效率。MTT法检测细胞增殖活性；划痕实验和Transwell小室实验检测细胞迁移、侵袭能力；Western Blot检测细胞中BCAN、G1/S-特异性周期蛋白-D1（Cyclin D1）、增殖细胞核抗原（PCNA）、基质金属蛋白酶2（MMP-2）、基质金属蛋白酶9（MMP-9）蛋白表达；双荧光素酶报告基因检测进一步验证miR-34a-5p与BCAN的靶向关系。结果胶质瘤组织中BCAN mRNA表达水平显著高于正常组织，而miR-34a-5p的表达水平显著低于正常组织，差异有统计学意义（均P＜0.05）；与人正常星形胶质细胞NHA相比，BCAN在人胶质瘤细胞中的表达显著升高（P＜0.05），而miR-34a-5p呈低表达（P＜0.05）。转染后，与NC组相比，miR-34a-5p过表达组细胞中miR-34a-5p水平显著升高（P＜0.05），BCAN mRNA和蛋白水平、细胞增殖率、划痕愈合率、穿膜细胞数以及Cyclin D1、PCNA、MMP-2、MMP-9的表达均显著降低（均P＜0.05）；在过表达miR-34a-5p的基础上，上调BCAN的表达可显著减弱miR-34a-5p过表达对U251 MG细胞增殖、迁移和侵袭的抑制作用（均P＜0.05）。双荧光素酶报告基因检测结果显示，miR-34a-5p的高表达可显著抑制BCAN-WT的荧光素酶活性（P＜0.05）。结论miR-34a-5p是BCAN的上游调控因子，过表达miR-34a-5p可靶向下调BCAN的表达，进而抑制胶质瘤细胞的增殖和迁移侵袭。

Abstract: ObjectiveTo explore the effects of overexpression of microRNA-34a-5p(miR-34a-5p) on the proliferation, migration and invasion of glioma cells, and to explore its targeting relationship with brevican(BCAN). MethodsThe brain glioma tissues of 36 patients with glioma treated in our hospital from October 2019 to October 2020 and the normal brain tissues of 36 patients who underwent internal decompression due to brain trauma were collected. Human glioma cells U373, U251 MG, LN229, A172 and human normal astrocytes NHA were cultured in vitro. reverse transcription quantitative PCR(RT-qPCR) was used to detect the expression levels of miR-34a-5p and BCAN mRNA in the tissues/cells. U251 MG cells in logarithmic growth phase were divided into control group(NC group), mimic-NC group(transfected with miR-34a-5p mimic negative control), miR-34a-5p overexpression group(transfected with miR-34a-5p mimic), miR-34a-5p overexpression+pcDNA3.1-NC group(co-transfected with miR-34a-5p mimic and pcDNA3.1-BCAN negative control empty vector plasmid), miR-34a-5p overexpression+ pcDNA3.1-BCAN group(co-transfected with miR-383-5p mimic and pcDNA3.1-BCAN). After 48 hours of transfection, the cells of each group were collected and the transfection efficiency was tested with RT-qPCR. the cell proliferation activity was detected with MTT method. The cell migration and invasion ability was detected with scratch test and Transwell chamber test. The protein expression of BCAN, G1/S-specific cyclin-D1(Cyclin D1), proliferating cell nuclear antigen(PCNA), matrix metalloproteinase 2(MMP-2) and matrix metalloproteinase 9(MMP-9) in cells was detected with Western Blot. The targeting relationship between miR-34a-5p and BCAN was further verified with dual luciferase reporter gene detection. ResultsThe expression level of BCAN mRNA in glioma tissues was significantly higher than that in normal tissues, while the expression level of mir-34a-5p was significantly lower than that in normal tissues(all P<0.05). Compared with human normal astrocytes NHA, the expression of BCAN in human glioma cells was significantly higher(P<0.05), while the expression of miR-34a-5p was low(P<0.05). After transfection, compared with the NC group, the miR-34a-5p level in the miR-34a-5p overexpression group was significantly increased(P<0.05), the levels of BCAN mRNA and protein, cell proliferation rate, scratch healing rate, number of transmembrane cells, and the expression of Cyclin D1, PCNA, MMP-2, and MMP-9 were significantly reduced(all P<0.05). On the basis of overexpression of miR-34a-5p, up-regulating the expression of BCAN could significantly reduce the inhibitory effect of overexpression of miR-34a-5p on the proliferation, migration and invasion of U251 MG cells(all P<0.05). The results of dual luciferase reporter gene detection showed that the high expression of miR-34a-5p could significantly inhibit the luciferase activity of BCAN-WT(P<0.05). ConclusionmiR-34a-5p is an upstream regulator of BCAN. Overexpression of miR-34a-5p can down regulate the expression of BCAN, and then inhibit the proliferation, migration and invasion of glioma cells.