miR-203靶定CAMTA1基因调控神经母细胞瘤细胞的迁移能力

MiR-203 regulates neuroblastoma cell migration by targeting CAMTA1

目的本研究旨在探讨miR-203在神经母细胞瘤组织中的表达水平及其在神经母细胞瘤细胞迁移过程中的作用机制。方法在神经母细胞瘤细胞中转染miR-203抑制剂，通过Transwell迁移实验研究其对迁移的影响。利用生物信息学技术预测miR-203的靶基因，并应用荧光报告基因检测、实时荧光定量PCR及WesternBlot对靶基因进行验证。采用siRNA干扰技术抑制靶基因CAMTA1的表达，共转染miR-203抑制剂及CAMTA1 siRNA，验证miR-203是否是通过靶基因影响细胞迁移。最后应用荧光定量PCR方法检测原发性和转移性神经母细胞瘤样本中miR-203的表达。结果神经母细胞瘤细胞系SH-SY-5Y和SK-N-SH中抑制miR-203表达后，与转染miR抑制剂对照相比，细胞迁移数目分别降低了41．34％和37．86％（P〈0．05）。miR-203能够直接靶向结合CAMTA1mRNA的野生型圳TR，转染miR-203抑制剂后，SH-SY-5Y和SK-N-SH细胞中内源性CAMTA1蛋白表达分别是转染miR阴性对照的2．13和1．78倍。（P〈0．05）。转染过表达CAMTA1质粒，SH-SY-5Y和SK-N-SH细胞迁移数目分别下降了25．27％和19．48％（P〈0．05）。在SH-SY5Y细胞中，同时共转miR-203抑制剂和CAMTA1 siRNA，与单纯转染miR-203抑制剂相比，细胞迁移能力增高了19．37％和（P〈0．05），与单纯转染抑制剂对照组相比，无统计学差异（P〈0．05）。与原发性神经母细胞瘤相比，在转移性神经母细胞瘤中，miR-203表达水平更高（P〈0．05）。结论miR-203与神经母细胞瘤样转移性相关，并且能够通过靶定CAMTAl基因影响神经母细胞瘤细胞的迁移能力。

Objective To explore the expression level of miR-203 in neuroblastoma tissue and elucidate its mechanism in neuroblastoma cell migration process. Methods In neuroblastoma cells transfected with miR-203 inhibitor, the impact of miR-203 upon migratory capacity was studied through Transwell assay. The bioinformatics was used to predict the target of miR-203. And the verification methods included luciferase reporter assay, real-time quantitative PCR and Western blot. The target gene CAMTA1 of miR-203 was confirmed. SiRNA interference technology was adopted to suppress the expression of target gene CAMTA1. Through co-transfections of miR-203 inhibitor and CAMTA1 siRNA, we determined whether miR-203 affected migration directly by targeting CAMTA1. And the expression of miR-203 was detected in metastatic and primary neuroblastoma specimens. Results Compared with ASO control, the numbers of migratory neuroblastoma cells SH- SY-SY and SK-N-SH decreased by 41.34% and 37. 860% after transfection with miR-203 ASO （P〈 0. 05）. MiR-203 could directly target CAMTA1 mRNA wild type 3＂UTR. After transfection with miR-203 ASO, the endogenous expression levels of CAMTA1 increased by 2. 13 and 1.78 folds in SH- SY-5Y and SK-N-SH cells compared with ASO control （P〈0. 05, P〈0. 05）. After transfection with CAMTA1 cDNA plasmid, the number of migration in SH-SY-fY and SK-N-SH decreased by 25.27% and 19. 480% respectively （P〈0. 05）. After co-transfections with miR-203 inhibitor and CAMTA1 siRNA, the number of migratory SH-SY5Y cell increased by 19. 37% compared with miR-203 inhibitor （P〈0. 05） and its number showed no significance compared with inhibitor control （P〈 0. 05）. MiR-203 had a higher expression level in metastatic neuroblastoma specimen than primary neuroblastoma specimen （P〈0. 05）. Conclusions MiR-203 is correlated with migratory capacity and it promotes the migration of neuroblastoma cell directly by targeting CAMTA1.