S100A4核定位在胰腺癌转移中的作用与机制研究

The role and mechanism of S100A4 nuclear localization in pancreatic cancer metastasis

目的:研究S100A4核定位在胰腺癌(pancreatic cancer,PC)转移中的作用与机制。方法:利用PC病程进展的组织芯片,通过免疫组化和HALO数字病理精准分析平台等技术,定量S100A4在组织细胞,特别是胞核中的表达情况,统计分析各检测指标与临床参数及生存期之间的关系。通过分子结构信息学分析、质粒构建与转染以构建基因调控的不同分组细胞研究模型;利用核质蛋白分离、免疫共沉淀、Western blot、划痕实验、Transwell实验、靶向剪切及转座酶技术(cleavage under targets and tagmentation,CUT&Tag)等研究S100A4核定位在PC转移中的作用与机制。结果:S100A4的高表达及其核定位与PC的T、N分期和不良预后正相关。PC细胞中S100A4核定位受小泛素相关修饰物(small ubiquitin-related modifier,SUMO)修饰调控,泛素结合酶9介导了PC细胞中S100A4的K22和K96位点与SUMO1结合,并通过SUMO特异性蛋白酶1实现去SUMO化,动态平衡PC细胞中S100A4的SUMO化修饰水平;调控S100A4蛋白的SUMO化修饰可改变PC细胞的体外转移能力;CUT&Tag测序结果证明S100A4核定位参与调控与肿瘤转移功能相关的基因网络。结论:S100A4核定位可提示PC预后不良,有望成为临床治疗方案制定的重要依据。发现阻断或抑制S100A4核定位的办法,可能成为抑制PC转移,特别是早期转移,改善PC患者预后的新靶点。

Objective:To explore the role and mechanism of S100A4 nuclear localization in pancreatic cancer(PC)metastasis.Methods:The expression of S100A4 in the PC cells,especially in the nucleus,was quantified by immunohistochemistry and HALO digital pathology precision analysis platform using the tissue microarray of PC.Statistical analysis was conducted to assess the correlations of each detection index with various clinical parameters and survival rates.Molecular structure informatic analysis,plasmid construction,and transfection were used to create different gene regulation cell research models.Techniques such as nuclear and cytoplasmic extraction,co-immunoprecipitation,Western blotting,wound healing assay,transwell assay,and cleavage under targets and tagmentation(CUT&Tag)assay were used to study the role and mechanism of S100A4 nuclear localization in PC metastasis.Results:High expression and the nuclear localization of S100A4 were positively correlated with T and N stages and poor prognosis in PC.The nuclear localization of S100A4 in PC cells was regulated by small ubiquitin-related modifier(SUMO)modification.Ubiquitin conjugating enzyme 9 mediated the binding of S100A4 to SUMO1 at K22 and K96 sites in PC cells,and deSUMOylation was achieved by sentrin-specific protease 1,dynamically balancing the SUMOylation level of S100A4.Regulating the SUMOylation of the S100A4 protein altered the metastatic ability of PC cells in vitro.The results of CUT&Tag sequencing confirmed that S100A4 nuclear localization was involved in the regulation of gene network related to tumor metastasis.Conclusion:S100A4 nuclear localization may indicate poor prognosis in PC and serve as a crucial basis for clinical decision making.Identifying methods to block or inhibit S100A4 nuclear localization may provide a new approach to suppress PC metastasis,especially the early metastasis,and improve the prognosis of PC patients.