环状RNA MAPK4通过吸附miR-125a-3p对神经胶质瘤细胞生物学行为的影响

Effects of circular RNA MAPK4 on biological behaviors of glioma cells by adsorbing miR-125a-3p

目的 探讨环状RNA MAPK4(circ-MAPK4)对神经胶质瘤细胞生物学行为的影响。方法 使用实时荧光定量聚合酶链式反应(qPCR)检测人神经胶质瘤细胞株(U138、U373、U87、A172、U251)和人正常性星形胶质细胞(HA1800)中circ-MAPK4、微小RNA-125a-3p(miR-125a-3p)表达水平,将U138细胞分为circ-MAPK4低表达组(si-circMAPK4组)、阴性对照组(si-NC组)、无转染组(control组),si-circMAPK4组、si-NC组U138细胞分别转染circ-MAPK4 siRNA及其阴性对照siRNA-NC,control组细胞不做转染处理;采用circNet、StarBase分析预测circ-MAPK4与miR-125a-3p的靶向关系并使用荧光素酶实验进行验证;将si-circMAPK4组U138细胞分为circ-MAPK4+miR-inhibitor组、circ-MAPK4+NC-inhibitor组2个亚组并完成相关转染,采用CCK-8法检测U138细胞活力、细胞集落实验检测细胞克隆形成率、Transwell检测细胞侵入细胞数目、流式细胞法检测细胞凋亡率,Western blot检测细胞增殖蛋白(Ki67、cycD)、侵袭蛋白(E-cadherin、N-cadherin、Vimentin)、凋亡蛋白(Ceaved-caspase3/caspase3)表达水平。结果 与HA1800细胞比较,U138、U373、U87和A172细胞的circ-MAPK4表达水平升高(P<0.05),miR-125a-3p表达水平下降(P<0.05);荧光素酶实验结果显示circ-MAPK4-mut/miR-125a-3p mimics、circ-MAPK4-mut/NC-mimics、circ-MAPK4-WT/NC-mimics细胞的荧光素酶相对活性均高于circ-MAPK4-WT/miR-125a-3p mimics细胞(F=437.659,P<0.001);与si-NC组比较,si-circMAPK4组细胞活力、克隆形成率、细胞侵入细胞数目、Ki67、cycD、N-cadherin和Vimentin蛋白表达降低(P<0.05),而细胞凋亡率、Ceaved-caspase3/caspase3和E-cadherin蛋白表达升高(P<0.05);与circ-MAPK4+NC-inhibitor组比较,circ-MAPK4+miR-inhibitor组细胞活力、克隆形成率、细胞侵入细胞数目、Ki67、cycD、N-cadherin和Vimentin蛋白表达升高(P<0.05),细胞凋亡率、Ceaved-caspase3/caspase3和E-cadherin蛋白表达降低(P<0.05)。结论 circ-MAPK4低表达能减弱人神经胶质瘤细胞U138细胞增殖和侵袭能力,促进细胞凋亡,可能与吸附miR-125a-3p作用有关。

Objective To investigate the effects of circular RNA MAPK4(circ-MAPK4)on biological behaviors of glioma cells.Methods The expression level of circ-MAPK4 and miR-125a-3p in human glioma cell lines(U138,U373,U87,A172,U251)and normal human astrocytes(HA1800)was detected by real-time fluorescence quantitative polymerase chain reaction(qPCR).U138 cells were divided into low-expression circ-MAPK4 group(si-circMAPK4 group),negative control group(si-NC group)and non-transfection group(control group).U138 cells in si-circMAPK4 group and si-NC group were transfected with circ-MAPK4 siRNA and its negative control SIRNA-NC,while cells in control group were not transfected.The targeted relationship between circ-MAPK4 and miR-125a-3p was predicted by circNet and StarBase analysis,which was verified by luciferase assay.U138 cells in si-circMAPK4 group were divided into two subgroups(circ-MAPK4+miR-inhibitor group,circ-MAPK4+NC-inhibitor group),and relevant transfection was completed.The activity of U138 cells was detected by CCK-8,formation rate of cell clone was detected by colony-forming assay,number of invasion cells was detected by Transwell assay,apoptosis rate was detected by flow cytometry,and expression levels of cell proliferation proteins(Ki67,cycD),invasion proteins(E-cadherin,N-cadherin,Vimentin)and apoptosis proteins(Ceaved-caspase3/caspase3)were detected by Western blot.Results Compared with HA1800 cells,expression level of circ-MAPK4 increased in U138,U373,U87,and A172 cells(P<0.05),while expression level of miR-125a-3p decreased(P<0.05).The results of luciferase assay showed that relative activities of luciferase in circ-MAPK4-mut/miR-125a-3p mimics,circ-MAPK4-mut/NC-mimics and circ-MAPK4-WT/NC-mimics cells were higher than those in circ-MAPK4-WT/miR-125a-3p mimics cells(F=437.659,P<0.001).Compared with si-NC group,cells activity,clone formation rate,number of invasion cells,expressions of Ki67,cycD,N-cadherin and Vimentin proteins decreased(P<0.05);while apoptosis rate,expressions of Ceved-caspase3/caspase3 and E-cadherin proteins increased in si-circMAPK4 group(P<0.05).Compared with circ-MAPK4+NC-inhibitor group,cells activity,clone formation rate,number of invasion cells,expressions of Ki67,cycD,N-cadherin,and Vimentin proteins increased(P<0.05);while apoptosis rate,expressions of Ceved-caspase3/caspase3 and E-cadherin proteins decreased in circ-MAPK4+miR-inhibitor group(P<0.05).Conclusion The low-expression circ-MAPK4 could weaken proliferation and invasion of human glioma U138 cells,and promote apoptosis,which might be related to the adsorption of miR-125a-3p.