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m6A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~6A Reader Protein IGF2BP1 on the Biological Behavior of Esophageal Squamous Cell Carcinoma Cells and Mechanism Exploration

    摘要
  • 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、 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。

     

    Abstract: 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~6A） 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~6A 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~6A modification sites. This study suggested that IGF2BP1 may interacted with other m~6A methyltransferases in an m~6A-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 exerted carcinogenic effects.

     

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