miR-92a-3p靶向调控PTEN对神经母细胞瘤细胞增殖、侵袭和迁移的影响

miR-92a-3P regulated the proliferation,invasion and migration of neuroblastoma cells by targeting PTEN

目的探讨微小RNA-92a-3p(micro RNA-92a-3p,miR-92a-3p)靶向调控PTEN对神经母细胞瘤(neuroblastoma,NB)细胞增殖、侵袭和迁移的影响。方法采用miR-92a-3p mimics及其抑制基因(inhibitor)分别转染至SH-SY5Y细胞,根据实验需要分为miR-92a-3p过表达组、NC过表达组、miR-92a-3p抑制组、NC抑制组,采用实时荧光定量聚合酶链反应(real time fluorescent quantitative polymerase chain reaction,qRT-PCR)检测转染后细胞内miR-92a-3p的表达水平;采用CCK-8检测实验、细胞克隆形成实验、细胞划痕实验、细胞侵袭实验检测miR-92a-3p对细胞增殖、侵袭和迁移能力的变化;采用基因软件预测miR-92a-3p的靶基因并用双荧光素酶报告体系进行验证;最后采用qRT-PCR和蛋白质印迹法检测过表达miR-92a-3p和抑制miR-92a-3p后对PTEN/PI3K/AKT信号通路相关分子的表达情况。结果qRT-PCR结果显示miR-92a-3p过表达组的表达含量(65.73±20.07)比miR-92a-3p抑制组的表达含量(0.33±0.02)显著增高,且差异有统计学意义(t=5.64,P=0.005)。CCK-8检测实验显示,miR-92a-3p过表达组能促进SH-SY5Y细胞的增殖活性(P均<0.001),miR-92a-3p抑制组则能有效抑制SH-SY5Y细胞的增殖活性(P=0.031,P=0.012,P<0.001,P<0.001)。细胞克隆形成实验结果显示,miR-92a-3p过表达组的细胞克隆数为(210±19)个,高于NC过表达组的细胞克隆数(144±5)个,组间比较差异有统计学意义(P=0.005);miR-92a-3p抑制组的细胞克隆数为(83±6)个,低于NC抑制组的细胞克隆数(137±13)个,组间比较差异有统计学意义(P=0.003)。细胞划痕实验及细胞侵袭实验结果显示,miR-92a-3p过表达组细胞迁移愈合率为(90.37±0.67)%,高于miR-92a-3p抑制组(41.03±0.56)%,组间比较差异有统计学意义(P<0.001);miR-92a-3p过表达组细胞穿膜数量为(106.80±9.28)个,高于miR-92a-3p抑制组(33.40±7.56)个,组间比较差异有统计学意义(P<0.001)。qRT-PCR和蛋白质印迹法结果显示,与NC过表达组相比,miR-92a-3p过表达组PTEN的mRNA表达量(0.43±0.02)和蛋白水平明显降低,而PI3K mRNA表达量(2.00±0.10)、AKT mRNA表达量(1.41±0.19)和各自蛋白水平明显升高,且差异均有统计学意义(P<0.001、P<0.001和P=0.028);miRNA-92a-3p抑制组可逆转上述作用,且差异均有统计学意义(P=0.043、P=0.046和P<0.001)。结论PTEN基因是miR-92a-3p的靶基因,miR-92a-3p可能通过促进NB细胞增殖、促进NB细胞侵袭、促进NB细胞迁移和抑制PTEN mRNA表达引发PTEN蛋白水平的降低。

Objective To explore the effects of microRNA-92a-3p(miR-92a-3p)on proliferation,invasion and migration of neuroblastoma(NB)cells and verify its effect on PTEN in NB cells and its regulatory mechanism.Methods miR-92a-3p mimics and its inhibitor were transfected into SH-SY5Y cells.According to experimental requirements,sh-SY5Y cells were divided into four groups of miR-92a-3p overexpression,NC overexpression,miR-92a-3p inhibition and NC inhibition.Real-time fluorescent quantitative polymerase chain reaction(qRT-PCR)was employed for detecting the expression level of miR-92a-3p in transfected cells.CCK-8,cell clonogenesis,cell scratch and cell invasion assays were utilized for detecting the changes of miR-92a-3p on cell proliferation,invasion and migration.The target genes of miR-92a-3p were predicted by gene software and verified by dual luciferase reporting system.Finally qRT-PCR and Western blot were used for detecting the expression of PTEN/PI3K/AKT signaling pathway related molecules after an overexpression of miR-92a-3p and an inhibition of miR-92a-3p.Results QRT-PCR revealed that the expression level of miR-92a-3p overexpressed group(65.73±20.07)was significantly higher than that of miR-92a-3p inhibited group(0.33±0.02)and the difference was statistically significant(t=5.64,P=0.005).CCK-8 assay showed that miR-92a-3p overexpression group could promote the proliferation activity of SH-SY5Y cells(all P<0.001),while miR-92a-3p inhibition group could effectively inhibit the proliferation activity of SH-SY5Y cells(P=0.031,P=0.012,P<0.001,P<0.001).The results of cell cloning formation experiment showed that the number of cell clones in miR-92a-3p overexpression group was(210±19),which was higher than that in NC overexpression group(144±5),and the difference between groups was statistically significant(P=0.005).The number of cell clones in miR-92a-3p inhibition group was(83±6),which was lower than that in NC inhibition group(137±13),and the difference was statistically significant(P=0.003).The assay results of cell scratch and cell invasion showed that the migration healing rate of miR-92a-3p group was(90.37±0.67)%and it was higher than that of inhibition group(41.03±0.56)%.The inter-group difference was statistically significant(P<0.001).The number of cells penetrating matrix glue in overexpressed miR-92a-3p group was(106.80±9.28).It was higher than that in inhibited group(33.40±7.56)and the difference was statistically significant(P<0.001).qRT-PCR and Western blot results showed that mRNA expression level and protein level of PTEN declined markedly in miR-92a-3p group.The expression levels of PI3K mRNA(2.00±0.10),AKT mRNA(1.41±0.19)and their protein levels spiked significantly with statistical significance(P<0.001,P<0.001,P=0.003).Inhibition of miR-92a-3p group reversed the above effects and the differences were statistically significant(P=0.043,P=0.046,P<0.001).Conclusions PTEN gene is a target gene of miR-92a-3p.And miR-92a-3p may lower PTEN protein level by promoting NB cell proliferation,NB cell invasion,NB cell migration and inhibiting PTEN mRNA expression.