miR-602调控神经母细胞瘤SH-SY5Y细胞生长、侵袭的功能研究

The function of miR-602 in regulating the growth and invasion of neuroblastoma in SH-SY5Y cells

目的探讨微小RNA-602(miR-602)对神经母细胞瘤(NB)SH-SY5Y细胞生长、侵袭的影响及其可能的机制。方法实时定量聚合酶链反应(qPCR)检测人NB细胞系SH-SY5Y和胚肾细胞系HEK-293中miR-602的表达水平。四甲基噻唑蓝(MTT)法检测miR-602对SH-SY5Y细胞生长的影响。Transwell侵袭实验检测miR-602对SH-SY5Y细胞侵袭的影响。筛选miR-602可能的靶基因,并通过荧光素酶报告基因系统结合qPCR技术验证miR-602对靶基因的调控作用。结果将健康者胚肾HEK-293细胞的表达量标化为1,人NB细胞系SH-SY5Y中miR-602的相对表达水平为3.83±0.85,差异有统计学意义(P<0.01);MTT实验结果显示,miR-602 mimics组细胞相对活性(1.20±0.05)高于NC mimics组(1.00±0.01),而miR-602 inhibitor组细胞相对活性为0.76±0.04低于NC inhibitor组(1.00±0.01),差异均有统计学意义(P<0.05);Transwell侵袭实验结果显示,miR-602 mimics组穿膜细胞数[(193.33±8.02)个]高于NC mimics组[(97.33±20.03)个],而miR-602 inhibitor组穿膜细胞数[(62.01±11.79)个]低于NC inhibitor组[(132.33±11.24)个],差异均有统计学意义(P<0.05);qPCR结果显示,在miR-602 mimics组重组人Sprouty相关EVH1域含蛋白1(SPRED1)mRNA表达水平较对照NC mimics组相对变化倍数为0.56±0.08,miR-602 inhibitor组SPRED1 mRNA表达水平较对照NC inhibitor组相对变化倍数为4.16±0.91,差异有统计学意义(P<0.01)。结论miR-602在SH-SY5Y细胞中高表达,可能通过调控SPRED1促进SH-SY5Y细胞生长和侵袭。

Objective To investigate the effects of microRNA-602(miR-602)on the growth and invasion of SH-SY5Y cell lines in neuroblastoma(NB)and its possible mechanism.Methods Real-time quantitative polymerase chain reaction(qPCR)was used to detect the expression of miR-602 in SH-SY5Y cell lines of NB and HEK-293 cells of normal human embryonic kidney.MTT asssy was used to detect the effect of miR-602 on the growth of SH-SY5Y cell lines.Transwell invasion assay was used to detect the effect of miR-602 on the invasion of SH-SY5Y cell lines of NB.The possible target genes of miR-602 were screened,and the regulatory effects of miR-602 on target genes were verified by the luciferase reporter gene system combined with qPCR technology.Results The expression level of HEK-293 cells in healthy people embryonic kidney was normalized as 1,the relative expression level of miR-602 in SH-SY5Y cell lines of NB was 3.83±0.85 and the difference was statistically significant(P<0.01).The results of MTT assay showed that the cell relative activity of miR-602 mimics group(1.20±0.05)was higher than that of NC mimics group(1.00±0.01),while the cell relative activity of miR-602 inhibitor group(0.76±0.04)was lower than that of NC inhibitor group(1.00±0.01),and the differences were statistically significant(P<0.05).The results of Transwell invasion experiment showed that the number of transmembrane cells in miR-602 mimics group(193.33±8.02)was higher than that in NC mimics group(97.33±20.03).The number of transmembrane cells of miR-602 inhibitor group(62.01±11.79)was lower than that of NC inhibitor group(132.33±11.24),and the differences were statistically significant(P<0.05).qPCR results showed that the expression level of recombinant human Sprouty-related EVH1 domain(SPRED1)mRNA in miR-602 mimics group was 0.56±0.08 compared with the control NC mimics group.The relative change factor of SPRED1 mRNA expression level in miR-602 inhibitor group was 4.16±0.91 compared with NC inhibitor group,and the differences were statistically significant(P<0.01).Conclusion miR-602 is highly expressed in SH-SY5Y cell lines,and it may promote the growth and invasion in SH-SY5Y cells by targeting the regulation of expression of SPRED1.