转染IGF2BP3基因siRNA和真核表达质粒的神经母细胞瘤细胞侵袭凋亡变化观察  

Changes in invasion and apoptosis of neuroblastoma cells transfected with IGF2BP3 gene siRNA and eukaryotic expression plasmid

作　　者：朱凯 王志茹[1] 高婷婷 吕志宝 ZHU Kai;WANG Zhiru;GAO Tingting;LYU Zhibao(Department of General Surgery,Shanghai Children’s Hospital,Shanghai 200062,China)

机构地区：[1]上海市儿童医院上海交通大学医学院附属儿童医院普外科,上海200062 [2]中国科学技术大学附属第一医院(安徽省立医院)儿外科

出　　处：《山东医药》2023年第27期1-6,共6页Shandong Medical Journal

基　　金：国家自然科学基金项目(82171696)。

摘　　要：目的观察转染胰岛素样生长因子2 mRNA结合蛋白3(IGF2BP3)基因siRNA和真核表达质粒的神经母细胞瘤(NB)细胞侵袭凋亡变化,以探讨IGF2BP3基因对NB细胞侵袭和凋亡的影响。方法选取NB细胞株IMR-32、SK-N-BE(2)、BE(2)-C、SH-SY-5Y,采用qRT-PCR检测IGF2BP3 mRNA、Western blotting法检测IGF2BP3蛋白、Transwell实验观察NB细胞的体外侵袭能力。根据上述实验结果,选择IGF2BP3表达水平最高的SK-N-BE(2)细胞及IGF2BP3表达水平最低的IMR-32细胞作为后续实验细胞。SK-N-BE(2)细胞转染针对IGF2BP3基因的小干扰RNA(siRNA)(SK-siIGF2BP3组),并设立细胞对照组(SK-Control组,只含有脂质体)和siRNA对照组(SK-siNC组,转染阴性对照序列)。IMR-32细胞转染IGF2BP3真核表达质粒(IMR-IGF2BP3组),并设立细胞对照组(IMR-Control组,只含有脂质体)和空载体对照组(IMR-Vector组,转染空载体)。转染成功后,采用qRT-PCR、Western blotting法和细胞免疫荧光染色法检测各组IGF2BP3 mRNA及蛋白,Transwell实验观察各组细胞侵袭能力,流式细胞术测算各组NB细胞凋亡率,Western blotting法检测各组细胞侵袭相关蛋白E-cadherin、N-cadherin、Vimentin。结果与SKControl组和SK-siNC组相比,SK-siIGF2BP3组IGF2BP3 mRNA、蛋白相对表达量降低,穿越Transwell小室膜细胞数减少,凋亡率升高(P均<0.05);与IMR-Control组和IMR-Vector组相比,IMR-IGF2BP3组IGF2BP3 mRNA、蛋白相对表达量升高,穿越Transwell小室膜细胞数增加,凋亡率降低(P均<0.05)。在SK-N-BE(2)细胞中,与SK-siNC组及SK-Control组比较,SK-siIGF2BP3组中N-cadherin、Vimentin蛋白相对表达量下降,E-cadherin蛋白相对表达量上升(P均<0.05);在IMR-32细胞中,与IMR-Vector组、IMR-Control组比较,IMR-IGF2BP3组N-cadherin和Vimentin蛋白相对表达量升高,E-cadherin蛋白相对表达量下降(P均<0.05)。结论转染IGF2BP3基因siRNA的NB细胞侵袭能力减弱、凋亡率增加,转染IGF2BP3真核表达质粒的NB细胞侵袭能力增强、凋亡率�Objective To observe the changes in invasion and apoptosis of neuroblastoma(NB)cells transfected with insulin-like growth factor 2 mRNA binding protein 3(IGF2BP3)gene siRNA and eukaryotic expression plasmid,in order to investigate the effects of IGF2BP3 gene on invasion and apoptosis of NB cells.Methods NB cell lines IMR-32,SK-N-BE(2),BE(2)-C,and SH-SY-5Y were selected.The protein and mRNA expression levels of IGF2BP3 were detect⁃ed by fluorescence quantitative PCR(qRT-qPCR)and Western blotting(WB),and Transwell assay was used to detect the invasive capacity of NB cell lines in vitro.SK-N-BE(2)cells with the highest expression level of IGF2BP3 and IMR-32 cells with the lowest expression level of IGF2BP3 were selected as the follow-up experiment cells.SK-N-BE(2)cells were transfected with siRNA targeting IGF2BP3 gene(SK-siIGF2BP3 group),and meanwhile,we set up a control group(SK-Control group,only liposomes)and a siRNA control group(SK-siNC group,transfected with the negative control se⁃quence).IMR-32 cells were transfected with IGF2BP3 eukaryotic expression plasmid(IMR-IGF2BP3 group),and mean⁃while,we set up a control group(IMR-Control group,only liposomes)and a empty vector control group(IMR-Vector group,transfected with the empty vector).After successful transfection,IGF2BP3 mRNA and protein of each group were detected by qRT-PCR,Western blotting,and immunofluorescence staining.The cell invasion ability of each group was ob⁃served by Transwell assay,and the apoptosis rate of NB cells in each group was measured by flow cytometry.The invasion-related proteins E-cadherin,N-cadherin and vimentin were detected by Western blotting.Results Compared with the SK-Control group and SK-siNC group,the relative expression levels of IGF2BP3 mRNA and protein decreased in the SK-si⁃IGF2BP3 group,the cells penetrating the Transwell membrane decreased,and the apoptosis rate increased(all P<0.05).Compared with the IMR-Control group and IMR-Vector group,the relative mRNA and protein expression levels of IGF2BP3 increased,

关 键 词：核糖核酸m6A甲基化读取器 胰岛素样生长因子2 mRNA结合蛋白家族 神经母细胞瘤 小干扰核糖核酸 细胞侵袭 细胞凋亡 

分 类 号：R739.4[医药卫生—肿瘤]