IGF2BP3在MNNG致人胃上皮细胞恶性转化中的作用及机制  

Role and mechanism of IGF2BP3 in malignant transformation of human gastric epithelial cells induced by MNNG

作　　者：任艺艺 杜丹丹 刘桐 尹立红 浦跃朴 梁戈玉 REN Yiyi;DU Dandan;LIU Tong;YIN Lihong;PU Yuepu;LIANG Geyu(Key Laboratory of Environmental Medicine Engineering,Ministry of Education/School of Public Health,Southeast University,Nanjing,Jiangsu 210009,China)

机构地区：[1]东南大学,公共卫生学院/环境医学工程教育部重点实验室,江苏南京210009

出　　处：《环境与职业医学》2022年第10期1146-1153,共8页Journal of Environmental and Occupational Medicine

基　　金：国家自然科学基金面上项目(81972998)。

摘　　要：[背景]N6-甲基腺苷(m6A)RNA甲基化修饰在环境致癌物诱发细胞恶性转化的过程中发挥着重要作用,但其作用及机制有待进一步探索。[目的]探究m6A结合蛋白中胰岛素样生长因子2 mRNA结合蛋白3(IGF2BP3)在N-甲基-N'-硝基-N-亚硝基胍(MNNG)致人胃黏膜上皮细胞GES-1恶性转化细胞中的作用及机制。[方法]基于MNNG致GES-1恶性转化细胞模型MC-30,应用转染慢病毒技术构建稳定敲低IGF2BP3的MC-30细胞株(MC30-shIGF2BP3,简写MC30-shI3),并设置阴性对照组(MC30-NC)。实时定量聚合酶链反应(qRT-PCR)和Western blotting实验分别检测IGF2BP3 mRNA和蛋白表达水平,RNA结合蛋白免疫沉淀技术(RIP-qPCR)验证恶转细胞MC-30中IGF2BP3蛋白与MYC mRNA的结合,放线菌素D实验检测MYC mRNA的稳定性。CCK-8、Transwell分别检测细胞增殖、迁移和侵袭能力,Western blotting实验检测EMT关键蛋白(N-cadherin、Vimentin、α-SMA、Snail)的表达。通过在MC30-shI3细胞中转染质粒过表达MYC的挽救实验,进一步观察细胞表型(增殖、迁移、侵袭)和EMT关键蛋白表达的变化来阐明下游靶基因MYC的作用。[结果]与对照组相比,5、10、20、40μmol·L–1 MNNG染毒GES-1细胞后,IGF2BP3 mRNA表达均上调(P<0.05)。20μmol·L–1 MNNG染毒GES-1细胞后,IGF2BP3 mRNA表达水平随染毒时间的延长呈上调趋势(P<0.05)。5μmol·L–1 MNNG恶转第10、20、30代IGF2BP3 mRNA和蛋白表达水平均上调(P<0.05)。qRT-PCR和Western blotting表明,与MC30-NC组相比,MC30-shI3组的细胞IGF2BP3 mRNA表达和蛋白表达水平明显下调(P<0.01);CCK8、Transwell表明,与MC30-NC组相比,MC30-shI3组的细胞增殖、迁移以及侵袭能力明显降低(P<0.01);Western blotting实验表明,与MC30-NC组相比,MC30-shI3组的EMT关键蛋白N-cadherin、Vimentin、α-SMA、Snail蛋白表达水平均明显下调(P<0.01);RIP-qPCR结果显示,在MC-30细胞中,与IgG组相比,IGF2BP3组中富集的MYC的mRNA水平更高(P<0.01);放线菌素D实验表明,[Background]N6-methyladenosine(m6A)RNA methylation may play an important role in the process of malignant transformation of cells induced by environmental carcinogens.However,the specific roles and mechanisms need to be further explored.[Objective]To explore the role and mechanism of m6A binding protein insulin-like growth factor 2 mRNA-binding protein 3(IGF2BP3)in the malignant transformation of human gastric mucosal epithelial cells GES-1 induced by N-methyl-N'-nitro-N-nitrosoguanidine(MNNG).[Methods]Based on the GES-1 malignant transformation cells MC-30,a stable knockdown IGF2BP3 MC-30 cell line(MC30-shIGF2BP3,abbreviated as MC30-shI3)was constructed by lentiviral transfection technology,and a negative control group(MC30-NC)was also prepared.Real-time quantitative polymerase chain reaction(qRT-PCR)and Western blotting were applied to detect the mRNA expression and protein levels of IGF2BP3.RNA binding protein immunoprecipitation(RIP-qPCR)was used to examine the combination between IGF2BP3 protein and MYC mRNA in malignant cells MC-30.Furthermore,the stability of MYC mRNA was detected by actinomycin D assay.CCK-8 and Transwell respectively were employed to detect cell proliferation,migration,and invasion.Western blotting was applied to detect the expression of EMT markers(N-cadherin,Vimentin,α-SMA,and Snail).The role of the downstream target gene MYC was further elucidated by a rescue assay in MC30-shI3 cells transfected with a plasmid overexpressing MYC to observe changes in cellular phenotypes(proliferation,migration,invasion)and expression of key EMT proteins.[Results]Compared with the control group,the expression of IGF2BP3 mRNA was up-regulated after 5,10,20,and 40μmol·L^(−1) MNNG infection of GES-1 cells(P<0.05).After 20μmol·L^(−1) MNNG infection,the expression level of IGF2BP3 mRNA increased with prolongation of exposure time(P<0.05).Compared with the control group,the mRNA and protein expression levels of IGF2BP3 were up-regulated in the 10th,20th,and 30th generations of 5μmol·L^(−1) MNNG

关 键 词：N-甲基-N'-硝基-N-亚硝基胍 胰岛素样生长因子2 mRNA结合蛋白3 GES-1细胞 恶性转化 上皮间质转化 

分 类 号：R735.2[医药卫生—肿瘤]