circ_0004771靶向miR-924调控胶质瘤U251细胞增殖、迁移和侵袭的机制研究

Study on the mechanism of circ_0004771 targeting miR-924 to regulate the proliferation,migration and invasion of glioma U251 cells

目的:探讨circ_0004771对胶质瘤U251细胞增殖、迁移和侵袭的影响及其分子机制。方法:选取33例胶质瘤组织标本以及正常脑组织,用实时荧光定量PCR(RT-qPCR)检测circ_0004771和miR-924的表达水平;将胶质瘤细胞株U251随机分为小干扰RNA阴性序列(si-NC)组(转染si-NC)、circ_0004771小干扰RNA(si-circ_0004771)组(转染si-circ_0004771)、模拟对照序列(miR-NC)组(转染miR-NC)、miR-924模拟物(miR-924)组(转染miR-924)、si-circ_0004771+抑制剂阴性序列(anti-miR-NC)组(共转染si-circ_0004771和anti-miR-NC)、si-circ_0004771+miR-924抑制剂(anti-miR-924)组(共转染si-circ_0004771和anti-miR-924);细胞计数试剂盒8(CCK-8)检测细胞活性;克隆形成实验检测细胞克隆形成数;划痕实验检测细胞划痕愈合率;Transwell小室实验检测细胞侵袭数;蛋白质印迹法检测E-cadherin和N-cadherin蛋白表达;双荧光素酶报告实验检测circ_0004771与miR-924的靶向关系。结果:与正常脑组织比较,胶质瘤组织中circ_0004771表达水平升高,miR-924表达水平降低(P<0.05)。与si-NC组(或miR-NC组)比较,si-circ_0004771组(或miR-924组)胶质瘤U251细胞活性、克隆形成数、划痕愈合率、侵袭数及细胞中N-cadherin蛋白表达均降低(P<0.05),E-cadherin蛋白表达升高(P<0.05)。circ_0004771靶向调控miR-924的表达。与si-circ_0004771和anti-miR-NC组比较,si-circ_0004771+anti-miR-924组胶质瘤U251细胞活性、克隆形成数、划痕愈合率、侵袭数及细胞中N-cadherin蛋白表达均升高(P<0.05),E-cadherin蛋白表达降低(P<0.05)。结论:下调circ_0004771表达可能通靶向上调miR-924抑制胶质瘤U251细胞的增殖、迁移和侵袭。

Objective:To explore the effect of circ_0004771 on the proliferation,migration and invasion of glioma U251 cells and its molecular mechanism.Methods:33 cases of glioma tissue specimens and normal brain tissues were selected,and the expression levels of circ_0004771 and miR-924 were detected by real-time fluorescent quantitative PCR(RT-qPCR).The glioma cell line U251 was randomly divided into small interfering RNA negative sequenc(si-NC)group(the cells were transfected with si-NC),circ_0004771 small interfering RNA(si-circ_0004771)group(the cells were transfected with si-circ_0004771),mimic control sequence(miR-NC)group(the cells were transfected with miR-NC),miR-924 mimic(miR-924)group(the cells were transfected with miR-924),si-circ_0004771+inhibitor negative sequence(anti-miR-NC)group(the cells were co-transfected with si-circ_0004771 and anti-miR-NC),si-circ_0004771+miR-924 inhibitor(anti-miR-924)group(the cells were co-transfected with si-circ_0004771 and anti-miR-924).And then cell viability was detected by cell counting kit 8(CCK-8).The number of cell clone formation was detected by clone formation experiment.The cell scratch healing rate was detected by scratch experiment.The number of cell invasion was detected by Transwell chamber.The protein expression of E-cadherin and N-cadherin were detected by Western blot method.The targeting relationship between circ_0004771 and miR-924 was detected by the dual luciferase reporter experiment.Results:Compared with normal brain tissue,the expression level of circ_0004771 in glioma tissue was increased,and the expression of miR-924 was decreased(P<0.05).Compared with the si-NC group(or miR-NC group),the activity of glioma U251 cells,clone formation number,scratch healing rate,invasion number and the expression of N-cadherin protein in si-circ_0004771 group(or miR-924 group)were decreased(P<0.05),but the expression of E-cadherin protein was increased(P<0.05).circ_0004771 targeted and regulated the expression of miR-924.Compared with the si-circ_0004771 and anti-miR-NC groups,the activity of glioma U251 cells,clone formation number,scratch healing rate,invasion number and the expression of N-cadherin protein in si-circ_0004771+anti-miR-924 group were increased(P<0.05),but the expression of E-cadherin protein was decreased(P<0.05).Conclusion:Down-regulating the expression of circ_0004771 may inhibit the proliferation,migration and invasion of glioma U251 cells by targeting up-regulation of miR-924.