摘要
目的探讨miR-20a-5p对人肾母细胞瘤细胞系WiT49裸鼠移植瘤的影响及作用机制。方法从GEO数据库下载基因表达芯片,应用GEO2R获得差异基因miR-20a-5p;通过cBioPortal数据库获得与miR-20a-5p表达具有正相关关系的NF-κB基因;通过targetscan数据库预测miR-20a-5p靶基因可能为NF-κB转录因子抑制蛋白家族的NFKBIB,并使用双荧光素酶实验进行验证;体外培养肾母细胞瘤细胞系WiT49,采用miR-20a-5p模拟物及其抑制基因构建慢病毒载体分别转染至WiT49细胞;将12只裸鼠随机分为3组,WiT49模型组、WiT49-miR-20a-5p过表达组、WiT49-miR-20a-5p敲低组;裸鼠成瘤实验检测各组瘤块质量及体积;应用实时荧光定量聚合酶链反应(qRT-PCR)检测各组瘤块中miR-20a-5p、NFKBIB及NF-κB表达情况;通过CCK-8细胞增殖实验验证各组瘤细胞增殖情况。结果miR-20a-5p在肾母细胞瘤中高表达,且与NF-κB表达呈正相关,miR-20a-5p与NFKBIB具有相互结合位点且存在结合作用;裸鼠成瘤实验中,WiT49-miR-20a-5p过表达组瘤块体积及质量较WiT49模型组均明显增加,差异有统计学意义(P<0.05);qRT-PCR实验中,WiT49-miR-20a-5p过表达组中miR-20a-5p及NF-κB表达均高于WiT49模型组,WiT49-miR-20a-5p过表达组中NFKBIB表达低于WiT49模型组,差异有统计学意义(P<0.05)。CCK-8细胞增殖实验中WiT49-miR-20a-5p过表达组细胞24及48 h吸光度高于WiT49模型组,WiT49-miR-20a-5p敲低组细胞24、48及72 h吸光度均低于WiT49模型组,差异有统计学意义(P<0.05)。结论miR-20a-5p可能是通过调控NFKBIB激活NF-κB通路促进人肾母细胞瘤细胞WiT49裸鼠移植瘤生长。
Objective To investigate the effect and mechanism of miR⁃20a⁃5p on human nephroblastoma cell line WiT49 transplanted tumor in nude mice.Methods The gene expression chip was downloaded from GEO database,and the differential gene miR⁃20a⁃5p was obtained by GEO2R.The NF⁃κB gene was positively correlated with the expression of miR⁃20a⁃5p through cBioPortal database.The target gene of miR⁃20a⁃5p was predicted to be NFKBIB of the NF⁃κB transcription factor suppressor protein family by targetscan database,and was verified by dual luciferase assay.Nephroblastoma cell line WiT49 was cultured in vitro and transfected into WiT49 cells with lentiviral vectors constructed with miR⁃20a⁃5p mimics and its suppressor gene.Twelve nude mice were randomly divided into three groups:WiT49 model group,WIT49⁃miR⁃20a⁃5p overexpression group and WIT49⁃miR⁃20a⁃5p knockdown group.The tumor mass and volume of each group were detected by tumor formation experiment in nude mice.real time fluorescent quantitative polymerase chain reaction(qRT⁃PCR)was used to detect the expression of miR⁃20a⁃5p,NFKBIB and NF⁃κB in each group;CCK⁃8 cell proliferation assay was used to verify the proliferation of tumor cells in each group.Results miR⁃20a⁃5p is highly expressed in nephroblastoma and is positively correlated with the expression of NF⁃κB.miR⁃20a⁃5p and NFKBIB have mutual binding sites and binding effects.In the tumor formation experiment of nude mice,the tumor volume and mass of WIT49⁃miR⁃20a⁃5P overexpression group were significantly increased compared with WiT49 model group,and the difference was statistically significant(P<0.05).In the qRT⁃PCR test,the expressions of miR⁃20a⁃5p and NF⁃κB in the WIT49⁃miR⁃20a⁃5p overexpression group were higher than those in the WiT49 model group,and NFKBIB expression in the WIT49⁃miR⁃20a⁃5p overexpression group was lower than that in the WiT49 model group,with statistical significance(P<0.05).CCK⁃8 cell proliferation assay sho
作者
王雅琦
李万富
阿依古再丽·麦麦江
艾尼娃·克然木
樊珈榕
梁鹏
娜菲沙·沙木西丁
WANG Yaqi;LI Wanfu;AYIGUZALI Maimaijiang;ANIWAR Kramer;FAN Jiarong;LIANG Peng;NAFISA Samusiddin(Department of Pediatric General Surgery,the First Affiliated Hospital of Xinjiang Medical University,Urumchi 830000,China)
出处
《实用医学杂志》
CAS
北大核心
2024年第4期490-495,共6页
The Journal of Practical Medicine
基金
自治区区域协同创新专项(科技援疆计划)(编号:2020E0267)。
