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TIM-ER2.0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IGF2BP1蛋白互作的其余N6-甲基腺嘌呤(N6-methyladenosine,m6A)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m6A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、Vimen-tin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m6A修饰位点.本研究提示,IGF2BP1可能通过m6A依赖的方式与其余m6A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 Effect of the m6A Reader Protein IGF2BP1 on the Biological Behavior of Esophageal Squamous Cell Carcinoma Cells and Mechanism Exploration
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 data-base,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 knock-down on epithelial-mesenchymal transition(EMT)-related proteins and phosphorylation levels of the PI3K/AKT pathway.The remai-ning N6-methyladenosine(m6A)methylases interacting with IGF2BP1 proteins were selected using the STRING,GEPIA databases and used for differential expression analysis.The downstream target genes and their m6A 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).Con-currently,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 posi-tive 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 m6A modification sites.This study suggested that IGF2BP1 may interacted with other m6A methyltransferases in an m6A-dependent manner to activate the phosphorylation of the PI3K/AKT pathway,thereby facilitated proliferation,migration,and invasion of ESCC cells.Additionally,it suppressed apoptosis and promoted EMT,thus exer-ted carcinogenic effects.
万方数据
