m^(6)A阅读蛋白IGF2BP1对食管鳞状细胞癌细胞生物学行为的影响及其机制探讨  

The Effect of the m^(6)A Reader Protein IGF2BP1 on the Biological Behavior of Esophageal Squamous Cell Carcinoma Cells and Mechanism Exploration

作　　者：孙姗 潘英杰 谭劲松 杨航 彭利红 冯刚 刘康 宋桂芹[1] SUN Shan;PAN Yingjie;TAN Jinsong;YANG Hang;PENG Lihong;FENG Gang;LIU Kang;SONG Guiqin(Institute of Basic Medicine and Forensic Medicine,North Sichuan Medical College,Nanchong,637100;Institute of Tissue Engineering and Stem Cells,Nanchong Central Hospital,The Second Clinical Medical College,North Sichuan Medical College,Nanchong,637000)

机构地区：[1]川北医学院基础医学与法医学研究所,南充637100 [2]南充市中心医院·川北医学院第二临床医学院组织工程与干细胞研究所,南充637100

出　　处：《基因组学与应用生物学》2024年第6期1078-1092,共15页Genomics and Applied Biology

基　　金：国家自然科学基金(82203851);四川省自然科学基金(2023NSFSC0731);四川省科技厅省院省校合作项目(2023YFSY0045)共同资助。

摘　　要：本研究旨在探讨胰岛素样生长因子-2 mRNA结合蛋白1(insulin like growth factor 2 mRNA binding protein 1,IGF2BP1)对食管鳞状细胞癌(esophageal squamous cell carcinoma,ESCC)细胞体外功能的影响及其作用机制。利用TIMER2.0数据库、免疫组化及实时定量PCR(real-time quantitative PCR,RT-qPCR)分析IGF2BP1在ESCC组织和癌旁组织中的蛋白及mRNA水平。采用siRNA敲低IGF2BP1在KYSE30和TE-1细胞中的表达,利用CCK-8实验、平板集落形成实验、划痕愈合实验、Transwell实验以及流式细胞术检测敲低IGF2BP1对细胞功能的影响。利用Western blot检测敲低IGF2BP1对上皮间质转化(epithelial-mesenchymal transition,EMT)相关蛋白及PI3K/AKT通路磷酸化水平的影响。利用STRING、GEPIA数据库筛选出与IGF2BPl蛋白互作的其余N^(6)-甲基腺嘌呤(N^(6)-methyladenosine,m^(6)A)甲基化酶并进行差异表达分析。通过RM2Target、SRAMP数据库预测下游靶基因及其m^(6)A修饰位点。结果显示,IGF2BP1在ESCC组织和细胞中高表达(P<0.05或P<0.001);敲低IGF2BP1后KYSE30和TE-1细胞增殖、迁移和侵袭能力减弱(P<0.05或P<0.001),细胞凋亡增多(P<0.05或P<0.001),同时促进E-cadherin的表达(P<0.05或P<0.01),抑制N-cadherin、Vimentin的表达(P<0.05或P<0.01)以及PI3K/AKT通路的磷酸化(P<0.05或P<0.01);锌指CCCH结构域蛋白13(zinc finger CCCH domain containing protein 13,ZC3H13)与IGF2BP1蛋白互作且在ESCC中的表达水平与IGF2BP1正相关;ZC3H13和IGF2BP1共同调控的潜在靶基因CNNM2、KIAA1549、SP3均具有高可信度的m^(6)A修饰位点。本研究提示,IGF2BP1可能通过m^(6)A依赖的方式与其余m^(6)A甲基化酶协调作用,激活PI3K/AKT通路的磷酸化而促进ESCC细胞的增殖、迁移、侵袭,抑制细胞凋亡,并促进EMT进程,发挥致癌作用。The objective of this study was to investigate the impact and underly mechanism of insulin-like growth factor 2 mRNA binding protein 1(IGF2BP1)on the in vitro functions of esophageal squamous cells(ESCC).We employed the TIMER2.0 database,immunohistochemistry,and real-time quantitative PCR(RT-qPCR)to examine the protein and mRNA levels of IGF2BP1 in esophageal cancer tissues and adjacent tissues.The expression of IGF2BP1 in KYSE30 and TE-1 cells was suppressed using siRNA,the impact of IGF2BP1 knockdown on cell function was assessed through CCK-8 assay,colony formation assay,scratch wound hea-ling assay,Trans-well assay,and flow cytometry.Western blot analysis was conducted to evaluate the influence of IGF2BP1 knockdown on epithelial-mesenchymal transition(EMT)-related proteins and phosphorylation levels of the PI3K/AKT pathway.The remaining N^(6)-methyladenosine(m^(6)A)methylases interacting with IGF2BPl proteins were selected using the STRING,GEPIA databases and used for differential expression analysis.The downstream target genes and their m^(6)A modification sites were predicted by the RM2Target and SRAMP databases.The findings revealed that IGF2BP1 was highly expressed in both ESCC tissues and cells(P<0.05 or P<0.001).After knocking down IGF2BP1,the proliferation,migration,and invasion capacities of KYSE30 and TE-1 cells were significantly attenuated(P<0.05 or P<0.001),while cellular apoptosis was markedly increased(P<0.05 or P<0.001).Concurrently,it facilitated the upregulation of E-cadherin expression(P<0.05 or P<0.01)and suppressed the expression of N-cadherin and Vimentin(P<0.05 or P<0.01),as well as phosphorylation of the PI3K/AKT signaling pathway(P<0.05 or P<0.01).Zinc finger CCCH domain containing protein 13(ZC3H13)interacted with the IGF2BP1 protein,and its expression level exhibited a positive correlation with that of IGF2BP1 in ESCC.The potential target genes CNNM2,KIAA1549,and SP3,which were co-regulated by ZC3H13 and IGF2BP1,all possessed highly credible m^(6)A modification sites.This study sugge

关 键 词：食管鳞状细胞癌 胰岛素样生长因子-2 mRNA结合蛋白1(IGF2BP1) N^(6)-甲基腺嘌呤(m^(6)A) 增殖 侵袭 凋亡 

分 类 号：R735.1[医药卫生—肿瘤]