摘要
目的神经母细胞瘤是儿童常见的恶性疾病，转移是致死的主要因素，其中微小RNA（microRNA，miRNA）在该疾病的转移中具有很重要的作用。本研究旨在探索miR-195是否通过RET调节神经母细胞瘤细胞的侵袭。方法在神经母细胞瘤细胞中转染mimicscontrol和miR-195mimics，建立过表达miR-195的细胞模型。通过Transwell侵袭实验研究过表达miR-195对侵袭的影响。利用生物信息学检测miR-195的靶基因，并应用荧光素酶报告基因鉴定其对靶基因31UTR的结合序列。实时定量PCR及western检测过表达miR-195对靶基因mRNA及蛋白的改变。应用siRNA干扰建立抑制靶基因的细胞模型，通过Transwell侵袭实验检测敲减靶基因对侵袭的影响。最后建立恢复靶基因表达的细胞模型，通过Transwell侵袭实验验证能力的恢复。结果在SH-SY5Y和SK-N-SH内，均成功过表达miR-195的相对水平分别为34±1．19和43±1．71，与对照组相比具有显著差异（P〈O．001）。SH—SY5Y过表达miR-195后发生侵袭的细胞数由93±2降至62±1，SK-N-SH过表达miR-195后发生侵袭的细胞数由82±2降至56±5，过表达miR-195后显著抑制细胞侵袭（P〈0．05）；生物信息学检测miR-195能够结合RETmRNA的3’UTR。过表达miR-195显著抑制RET的mRNA和蛋白质水平（P〈O．05）。过表达miR-195显著抑制报告质粒RET3’UTRwT的荧光素酶的相对活性（P〈O．05）。RNA干扰均抑制两种细胞内RET（一个在转化中发生重排的原癌基因，RET原癌基因）的mRNA和蛋白的水平（P〈0．05）。在SH-SY5Y内，敲减RET后发生侵袭的细胞数目由89±2降至62±5，在SK-N-SH中由78±2降至63±1，均具有显著差异（P〈0．05）。在恢复实验中，对照组和实验组发生侵袭的数目分别为84±3、64±9、65±5和82±1，恢复RET表达恢复细胞的侵袭能力。结论miR-195与神经母细胞瘤侵袭能力相关，并且能够通过靶向RET调节神经母细胞瘤细胞的侵袭。
Objective To detect whether miR-195 regulates neuroblastoma ceil invasion by targeting RET. Methods A miR-195-overexpressing cell model was established by transfecting cells with mimics control and miR-195 mimics. Transwell invasion assay was performed for detecting cell invasion activity. Bioinformatics was used to predict the target of miR-195, together with luciferase reporter assay. Real-time quantitative polymerase chain reaction （PCR） and Western blot were performed for detecting the target gene expression. Then RET-inhibiting cell model was established by RNAi interference and used for examining cell invasion activity. And the expression of RET was restored and the invasion ability detected. Results The expression of miR-195 was enhanced by 34 ± 1.19 and 43 ± 1.71 folds in SH-SYSY and SK-N-SH respectively. They showed significant difference compared with control group （P〈0. 05）. An overexpresion of miR-195 decreased cell invasion count from 93 ±2 to 62 ± 1 in SH-SY5Y and from 82 ± 2 to 56 ± 5 in SK-N-SH. An overexpression of miR- 195 inhibited cell invasion significantly （P〈0. 05）. Bioinformatics indicated that miR-195 could bind to 3＇UTR of RET mRNA. An over-expression of miR-195 could significantly inhibit the mRNA and protein expressions of RET （P〈0. 05）. And an overexpression of miR-195 could also inhibit significantly the relative luciferase activity of RET 3＇ UTR WT （P〈0. 05）. A knockdown of RET decreased cell invasion count from 89 ± 2 to 62 ± 5 in SH-SY5Y and from 78 ± 2 to 63 ± 1 in SK-N-SH. A knockdown of RET inhibited significantly cell invasion （P〈0. 05）. In rescue assay, invasion cell counts of control and experimental groups were 84 ± 3,64 ± 9,65 ± 5 and 82 ± 1 respectively. Restored expression of RET rescued cell invasion activity. Conclusions Correlated with neuroblastoma cell invasion,MiR-195 regulates neuroblastoma cell invasion by targeting RET.
出处
《中华小儿外科杂志》
CSCD
2015年第10期775-779,共5页
Chinese Journal of Pediatric Surgery
基金
国家自然科学基金（81272986）
山东省自然科学基金重点项目（ZR2011HZ002）
山东省教育厅课题（J11LF58）
关键词
神经母细胞瘤
侵袭
转染
Neuroblastoma
Invasion
Transfection
