微小RNA-34对小儿恶性胶质瘤细胞增殖、迁移和侵袭的影响

Effect of microRNA-34 on proliferation,migration and invasion of malignant glioma cells in children

目的观察微小RNA(miR)-34对小儿恶性胶质瘤细胞增殖、侵袭和转移的影响。方法选取2017年6月到2019年6月我院收治的49例恶性胶质瘤组织和癌旁组织作为研究对象,采用荧光定量聚合酶链反应(PCR)分析肿瘤组织和癌旁组织miR-34表达水平。用过表达对照和miR-34慢病毒感染U251神经胶质瘤细胞,建立对照组和miR-34组稳定细胞系。采用细胞计数试剂盒8(CCK-8)、划痕实验和Transwell实验分析对照组和miR-34组细胞增殖、迁移和侵袭能力。采用生物信息学、双荧光素酶报告基因分析miR-34靶基因。蛋白质印迹法(Western blot)分析靶基因表达水平。计量数据比较采用t检验。结果神经胶质瘤组织中miR-34表达水平(0.44±0.10)明显低于癌旁组织(1.14±0.15),差异有统计学意义(t=4.109,P<0.05)。对照组细胞吸光度值(2.09±0.31)明显高于miR-34组(1.63±0.27),差异有统计学意义(t=3.016,P<0.05)。对照组细胞划痕愈合率[(89.37±8.19)%]明显高于miR-34组[(58.32±6.82)%],差异有统计学意义(t=4.781,P<0.05)。对照组细胞迁移数量[(136.22±15.29)个]明显高于miR-34组[(76.29±9.29)个],差异有统计学意义(t=5.891,P<0.05)。生物信息学和双荧光素酶报告基因结果显示SOX4基因是miR-34的靶基因。癌旁组织中SOX4蛋白水平(0.91±0.12)明显低于肿瘤组织(2.86±0.16),差异有统计学意义(t=3.471,P<0.05)。对照组细胞SOX4蛋白表达水平(1.28±0.14)明显高于miR-34组(0.64±0.12),差异有统计学意义(t=3.003,P<0.05)。对照组细胞TMEM2蛋白表达水平(1.37±0.17)明显高于miR-34组(0.76±0.11),差异有统计学意义(t=2.409,P<0.05)。结论 miR-34在恶性胶质瘤组织中呈低表达,通过调控SOX4蛋白表达水平,调节恶性神经胶质瘤细胞的增殖、侵袭和转移。

Objective To investigate the effect of microRNA(miR)-34 on proliferation,migration and invasion of malignant glioma cells in children.Methods Totally,49 cases of malignant glioma tissues and paracancerous tissues in our hospital from June 2017 to June 2019 were selected as the research objects.The expression levels of miR-34 in tumor tissues and adjacent tissues were analyzed by fluorescence quantitative polymerase chain reaction(PCR).U251 glioma cells were infected with over-expression control and miR-34 lentivirus to establish stable cell lines(control group and miR-34 group).The cell proliferation,migration and invasion ability in control group and miR-34 group were analyzed by cell counting kit 8(CCK-8)assay,scratch test and Transwell test.The relationship between miR-34 and its targeting gene was analyzed by bioinformatics and dual luciferase reporter genes.The expression of targeting gene was analyzed by Western blotting.The measurement data were compared by t-test.Results The expression level of miR-34 in glioma tissues(0.44±0.10)was significantly lower than that in adjacent tissues(1.14±0.15,t=4.109,P<0.05).The absorbance value of control group(2.09±0.31)was significantly higher than that of miR-34 group(1.63±0.27,t=3.016,P<0.05).The wound healing rate of control group[(89.37±8.19)%]was significantly higher than that of miR-34 group[(58.32±6.82)%,t=4.781,P<0.05].The number of migrating cells in control group[(136.22±15.29)]was significantly greater than that in miR-34 group[(76.29±9.29),t=5.891,P<0.05].Bioinformatics and dual luciferase reporter gene results showed that SOX4 gene was the target gene of miR-34.The expression level of SOX4 protein in adjacent tissues(0.91±0.12)was significantly lower than that in tumor tissues(2.86±0.16,t=3.471,P<0.05).The expression level of SOX4 protein in control group(1.28±0.14)was significantly higher than that in miR-34 group(0.64±0.12,t=3.003,P<0.05).The expression level of TMEM2 protein in control group(1.37±0.17)was significantly higher than that in miR-34 group(0.76±0.11,t=2.409,P<0.05).Conclusion miR-34 has low expression in gliomas.It can regulate the proliferation,migration and invasion of gliomas by regulating the expression of SOX4 protein.