miR-93对神经母细胞瘤细胞侵袭和迁移的影响及机制研究  

Effects of miR-93 on invasion and migration in neuroblastoma cell and its mechanism

作　　者：陈鑫[1] 韩芦芦[2] 姜忠[1] 郝希伟[1] 段于河[1] 张虹[1] 董蒨[1] 

机构地区：[1]青岛大学附属医院小儿外科,266003 [2]青岛大学附属医院手术室,266003

出　　处：《中华小儿外科杂志》2015年第12期930-935,共6页Chinese Journal of Pediatric Surgery

基　　金：国家自然科学基金面上项目（81272986）;山东省自然科学基金重点项目（ZR2011HZ002）;山东省教育厅课题（J11LF58）

摘　　要：目的研究miR-93对神经母细胞瘤细胞侵袭和迁移能力的影响及可能的分子机制。方法收集13例确诊的神经母细胞瘤患儿原发部位和转移部位组织样本，利用实时定量PCR的方法检测样本中miR-93的表达水平。在神经母细胞瘤细胞SH—SY5Y和SH—N-SH细胞中转染miR-93mimic，转染24h后利用实时定量PCR技术检测SH—SY5Y和SH—N-SH中miR-93的表达水平。利用Transwell侵袭实验检测过表达miR-93后对细胞侵袭能力的影响。利用体外划痕实验检测过表达miR-93对细胞迁移能力的影响。利用Western blot方法检测过表达miR-93mimic后SH—SY5Y和SH-N-SH细胞中MYCN蛋白表达水平。在Targetscan中找出可能与miR-93作用的MYCN的3’UTR序列，设计合成野生型与突变型MYCN的3’UTR的PCR引物，构建野生型与突变型荧光素酶报告载体，用X-tremeGENE将荧光素酶报告载体分别与miR-93mimic共转染到SH—SY5Y细胞中，用双荧光素酶检测试剂盒检测荧光素酶活性，验证miR-93对MYCN的靶向调控作用。结果与原发性神经母细胞瘤相比，miR-93在转移瘤中表达显著降低。过表达miR-93后SH—SY5Y和SH—N-SH细胞侵袭和迁移能力显著降低。过表达miR-93后，SH-SYSY和SH-N-SH细胞内MYCN蛋白表达下调。荧光素酶活性检测表明miR-93能够抑制MYCN的荧光素酶活性。结论MiR-93能够通过负调控MYCN而抑制神经母细胞瘤细胞侵袭和迁移能力。Objective To explore the effects of miR-93 on cell invasion and migratory capacities in neuroblastoma （NB） cell and its mechanism. Methods The expression levels of miR-93 were detected by quantitative reverse transcription-polymerase chain reaction （qRT-PCR） in primary and metastatic neublastoma tissues from 13 diagnosed children patients, miR-93 was transfected into SH-SYSY and SH-N-SH neuroblastoma cells and its expression level detected by qRT-PCR. The invasive capacities of SH-SY5Y and SH-N-SH ceils were examined by Transwell invasion after an over-expression of miR-93. And the migratory capacities of SH-SYSY and SH-N-SH cell were examined by wound healing assays after an over-expression of miR-93. And the expression levels of MYCN in SH-SY5Y and SH-N-SH cells were detected by Western blot. The potential miR-93- targeting MYCN 3 ＇UTR sequence in Targetscan, primers of wild and mutant type of MYCN 3＂ UTR, were designed and synthesized for cloning 3＇UTR of MYCN into luciferase reporter vector. And lueiferase activity was detected and verified by double luciferase detection kit after co-transfecting lueiferase reporter vector and miR-93 into SH-SYSY ceils with X-tremeGENE. Results miR-93 became down-regulated in metastatic neuroblastoma tissues compared with primary tissues. And the invasive and migratory capacities decreased after trans{ecting miR-93 in SH-SY5Y and SH-N-SH cells. And the expression level of MYCN decreased after transfecting miR93. MiR-93 could suppress the enzymatic activity of luciferase reporter vector of MYCN. Conclusions MiR-93 can suppress the invasion and migration of NB cells by down-regulating the expression of MYCN.

关 键 词：神经母细胞瘤 肿瘤转移 荧光素 

分 类 号：R739.41[医药卫生—肿瘤]