微小RNA-135b靶向结合叉头蛋白N3激活磷脂酰肌醇-3激酶/蛋白激酶B通路促进神经母细胞瘤增殖的研究

MicroRNA-135b promotes the proliferation of neuroblastoma cells by targeting forkhead box N3 and activating the phosphatidylinositol 3 kinase/protein kinase B signaling pathway

目的探究微小RNA-135b(miR-135b)对人神经母细胞瘤SH-SY5Y细胞增殖的影响及其分子机制.方法用简单随机法将SH-SY5Y细胞分为miR-135b模拟物(mimics)组和miR-135b抑制物(inhibitor)组,以及阴性对照NC mimics组和NC inhibitor组并转染,通过实时荧光定量聚合酶链反应(RT-qPCR)方法检测各组细胞转染效果,细胞划痕实验、细胞活力检测(CCK-8)、细胞侵袭实验(Transwell™)检测细胞迁移、增殖和侵袭的生物学功能.生物信息学软件预测miR-135b的下游靶基因,双荧光素酶报告实验加以验证.免疫印迹法(Western blot)检测各组叉头蛋白N3(FOXN3)和磷脂酰肌醇-3激酶/蛋白激酶B(PI3K/Akt)通路相关蛋白的表达情况.组间比较用t检验,多组比较用单因素方差分析.结果miR-135b mimics组的细胞迁移、增殖和侵袭能力高于NC mimics组[(28.47±1.10)%比(15.04±1.38)%,t=10.70,P<0.01;1.97±0.11比1.47±0.10,t=5.79,P<0.05;265.00±21.93比204.56±29.54,t=2.85,P<0.05];miR-135b inhibitor组的细胞迁移、增殖和侵袭能力低于NC inhibitor组[(7.67±1.24)%比(14.06±2.92)%,t=2.80,P<0.05;1.04±0.08比1.49±0.12,t=5.39,P<0.05;75.89±3.00比201.11±19.78,t=10.84,P<0.01].生物信息学软件预测发现FOXN3是miR-135b的靶基因之一,双荧光素酶报告基因实验证实.Western blot示miR-135b mimics组的FOXN3表达低于NC mimics组(0.35±0.15比0.82±0.07,t=4.96,P<0.01),PI3K、p-Akt/Akt表达高于NC mimics组(1.50±0.18比0.85±0.05,t=5.31,P<0.01;1.19±0.08比0.89±0.02,t=5.53,P<0.01);miR-135b inhibitor组的FOXN3表达高于NC inhibitor组(1.97±0.09比0.75±0.10,t=2.78,P<0.05),PI3K、p-Akt/Akt表达低于NC inhibitor组(0.77±0.11比1.03±0.07,t=4.58,P<0.05;0.64±0.01比0.82±0.07,t=4.43,P<0.05).结论miR-135b可能通过靶向结合FOXN3激活PI3K/Akt信号通路促进人神经母细胞瘤细胞增殖.

Objective To explore the effect of microRNA-135b(miR-135b)on the proliferation of neuroblastoma SH-SY5Y cells and its molecular mechanisms.Methods SH-SY5Y cells were transfected with miR-135b mimics,miR-135b inhibitor,and negative controls NC mimics,NC inhibitor,and grouped by simple randomization.The expression of miR-135b was detected by real-time quantitative polymerase chain reaction(RT-qPCR).Biological functions of miR-135b in migration,proliferation and invasion were examined by cell scratch,cell counting kit-8(CCK-8)and cell invasion assays.The bioinformatics soft-wares were used to predict target genes of miR-135b and luciferase reporter gene detection experiments were performed to verify it.The expression of phosphatidylinositol 3 kinase/protein kinase B(PI3K/Akt)signa-ling pathway and forhead box N3(FOXN3)protein was detected by Western blotting.T-test was used for inter group comparisons,and one-way ANOVA was used for inter group comparisons.Results The migra-tion,proliferation and invasion abilities in miR-135b mimics group were greater than those in NC mimics group[(28.47±1.10)%vs.(15.04±1.38)%,t=10.70,P<0.01;1.97±0.11 vs.1.47±0.10,t=5.79,P<0.05;265.00±21.93 vs.204.56±29.54,t=2.85,P<0.05].The migration,prolifera-tion and invasion abilities in miR-135b inhibitor group were lower than those in NC inhibitor group[(7.67±1.24)%vs.(14.06±2.92)%,t=2.80,P<0.05;1.04±0.08vs.1.49±0.12,t=5.39,P<0.05;75.89±3.00 vs.201.11±19.78,t=10.84,P<0.01].Bioinformatics softwares predicted FOXN3 was one of the target genes of miR-135b,which was confirmed by luciferase reporter assay.West-em blotting showed that FOXN3 expression was lower in the miR-135b mimics group than in the NC mimics group(0.35±0.15 vs.0.82±0.07,t=7.01,P<0.05),and the expression of PI3K and p-Akt/Akt ra-tio were higher in the miR-135b mimics group than those in the NC mimics group(1.50±0.18 vs.0.85±0.05,t=4.97,P<0.05;1.19±0.08 vs.0.89±0.02,t=5.54,P<0.05);FOXN3 expression was higher in the miR-135b inhibitor group than in the NC inhibitor group(0.97±0.09 vs.0.75±0.10,t=3.87,P<0.05),and the expression of PI3K and p-Akt/Akt ratio were lower in the miR-135b inhibitor group than those in the NC inhibitor group(0.77±0.11 vs.1.03±0.07,t=2.94,P<0.05;0.64±0.01 vs.0.82±0.07,t=3.47,P<0.05).Conclusion MiR-135b may target FOXN3 and activate PI3K/Akt signaling pathway to promote the proliferation of neuroblastoma cells.