miR-144通过靶定ITGβ1调控神经母细胞瘤的侵袭转移  

miR-144 regulates the invasion and metastasis of neuroblastoma by target ITGβ1

作　　者：吴翮 徐升 公红磊 韩芦芦[2] 陈鑫[3] 

机构地区：[1]青岛市妇女儿童医院小儿普外科,266000 [2]青岛大学附属医院手术室,266003 [3]青岛大学附属医院小儿外科,266003

出　　处：《中华小儿外科杂志》2017年第4期260-264,共5页Chinese Journal of Pediatric Surgery

基　　金：国家自然科学基金面上项目（81272986）;国家自然科学基金青年基金项目（81502363、81602395）;山东省自然科学基金重点项目（ZR2011HZ002）

摘　　要：目的 本研究旨在探讨miR-144对神经母细胞瘤的侵袭和转移的影响,并探索miR-144是否通过靶定ITGβ1调节神经母细胞瘤的侵袭和转移.方法 利用生物信息学技术预测miR-144的靶基因ITGβ1,应用荧光报告基因检测以及荧光定量PCR、Western Blot实验对靶基因进行验证.在神经母细胞瘤SH-SY5Y和SK-N-BE分别瞬时转染化学合成的成熟双链miR-144和靶基因敲降质粒及对照质粒,建立过表达miR-144细胞模型和靶基因敲降细胞模型.通过 Transwell侵袭实验和划痕实验检测细胞的体外侵袭和转移能力.结果 Transwell实验结果表明,在神经母细胞瘤细胞SH-SY5Y和SK-N-BE中过表达miR-144后,细胞的侵袭数目分别为41±2和55±2,与对照组相比差异有统计学意义（P〈0.01）.抑制ITGβ1基因的表达后,体外划痕实验结果表明,过表达miR-144后,与对照组相比,SH-SY5Y和SK-N-BE细胞的转移能力分别下降了65.45%和75.41%（P〈0.01）.荧光报告基因检测结果显示过表达miR-144后,含有结合位点的荧光报告质粒的相对荧光量降低了37.11%（P〈0.01）,而将结合位点进行基因突变后miR-144对靶基因的抑制作用消失.过表达miR-144后,与对照组相比,SH-SY5Y和SK-N-BE细胞内的ITGβ1 mRNA水平分别降低了40%和43%,Western Blot检测结果显示,ITGβ1蛋白水平分别降低了39%和40%.结论 miR-144通过靶定ITGβ1基因抑制神经母细胞瘤的侵袭和转移过程.Objective To explore the effects of miR-144 on the invasion and metastasis of metastatic neuroblastoma by targeting ITGβ1.Methods Bioinformatic techniques were employed for predicting the target gene ITG beta 1 of miR-144.And fluorescent reporter gene detection, real time polymerase chain reaction （PCR） and Western blot were used to verify the target gene.SH-SY5Y and SK-N-BE were transiently transfected into miR-144 mimics, target gene knockout plasmid and control plasmid respectively.The cell models of over-expressed miR-144 and target gene knockdown were established.And the invasion and metastasis of cells were detected by Transwell and scratch assays.Results After an over-expression of miR-144 in SH-SY5Y and SK-N-BE neuroblastoma cells, the number of invasive cells was 41±2 and 55±2 respectively.As compared with control group, the difference was significant （P〈0.01）.After inhibiting the expression of ITGβ1 gene, the number of invasion were 40±5 and 55±5 in SH-SY5Y and SK-N-BE cells respectively.As compared with control group, the differences were significant （P〈0.01）.After an over-expression of miR-144, the metastatic capacities of SH-SY5Y and SK-N-BE cells decreased by 65.45% and 75.41% respectively （P〈0.01）.As shown by the results of luciferase detection, after an over-expression of mir-144, the relative fluorescent amount of fluorescence report plasmid containing binding sites decreased significantly by 37.11% （P〈0.01）, the inhibitory effect of miR-144 on target gene disappeared after gene mutation.As compared with control group, the levels of mRNA and ITGβ1 decreased by 40% and 43% in SH-SY5Y and SK-N-BE cells respectively.And the protein levels of miR-144 and ITGβ1 decreased by 39% and 40% respectively.Conclusions miR-144 inhibits the invasion and metastasis of neuroblastoma by targeting ITGβ1 gene.

关 键 词：神经母细胞瘤 侵袭 转移 

分 类 号：R739.4[医药卫生—肿瘤]