14-3-3ζ经上皮-间质细胞转化诱导胆管癌细胞骨桥蛋白异常糖基化的机制研究

Cholangiocarcinoma (CCA) is a malignant tumor with high invasion potential. Researches have indicated that epithelial-mesenchymal transition (EMT) is an initiation step and a core aspect in the invasion and metastasis of tumor. So it is great significance to screen early diagnosis markers and seek effective therapeutic targets for CCA. In previous works, the applicant has successfully established the EMT models of CCA, and verified that 14-3-3ζ is a key regulatory molecule of EMT. Meanwhile, we have preliminarily detected the differential profiling of osteopontin (OPN) after EMT by lectin microarray, and revealed that 14-3-3ζ was closely correlated with the aberrant glycosylation of OPN. Base on these experimental data, this project intends to investigate the pathological significance of 14-3-3ζ and OPN in invasion and metastasis of CCA through clinical characteristics and follow-up findings. And then, lectin microarray combined with mass spectrometry will be employed to detect the differential glycan profiling of OPN in 14-3-3ζ induced the EMT model, and screen the glycosylation site and the carbohydrate-processing enzymes. Furthermore, the effect of OPN aberrant glycosylation in invasion and metastasis of CCA will be investigated using molecular and cellular biology methods, such as small molecule interference, and the experiments in vivo and in vitro. Finally, the molecular mechanism of 14-3-3ζ regulating OPN aberrant glycosylation via EMT will be explored. The project is expected to provide the scientific basis for elucidating the molecular mechanisms of invasion and metastasis, and new ideas for the therapeutic strategy of CCA.

胆管癌(CCA)具有高侵袭性的恶性肿瘤。研究表明，上皮-间质细胞转化(EMT)是侵袭转移的起始步骤，对筛选CCA早期诊断标志物和高效治疗靶点具有重大意义。申请者前期成功构建了CCA细胞EMT模型，证实14-3-3ζ是调控EMT的核心分子，并初步检测了癌细胞EMT转化后骨桥蛋白(OPN)特异性糖链谱，发现14-3-3ζ与OPN异常糖基化密切相关。本项目拟在前期工作基础上，结合临床资料明确14-3-3ζ和OPN在CCA中的病理学意义；通过凝集素芯片联合质谱技术检测经14-3-3ζ介导EMT转化后OPN特异性糖谱，筛查OPN异常糖基化位点和糖链加工酶；采用小分子干扰等分子细胞生物学方法和体内外实验，探讨OPN异常糖基化对CCA细胞侵袭转移的影响，并进一步探讨14-3-3ζ经EMT诱导OPN异常糖基化的分子机制。本项目实施为阐明CCA早期侵袭转移的机制提供科学依据，为寻找高效治疗靶点提供新思路。

胆管癌因其高侵袭性和极差的预后而成为目前肿瘤学研究中的难点问题。研究表明，上皮-间质细胞转化（EMT）是恶性肿瘤细胞侵袭转移的核心环节和起始步骤，具有启动多种生物学过程的特性，共同促进肿瘤的侵袭转移。本课题（1）检测14-3-3ζ和OPN在胆管癌中的表达均呈高表达（P<0.05），两者呈正相关(γ=0.5626，P<0.0001)，结合临床病理资料，提示二者的表达与胆管癌的病理类型、分期、侵袭转移和预后密切相关(P<0.05)。（2）构建14-3-3ζ+/-胆管癌细胞株EMT体外模型，靶向下调14-3-3ζ，OPN的表达受到抑制(P<0.05)，同时，体内、外实验表明癌细胞的侵袭移动能力亦受到抑制(P<0.05)。（3）运用凝集素芯片技术分析提示OPN特异性糖链谱，筛选具有显著差异的凝集素LBA，其特异性糖链GalNAc(α1,3)Galβ，特异性催化糖基转移酶多肽-N-乙酰半乳糖胺转移酶（GalNAc-T），其家族中GalNAc-T3与糖蛋白O-糖基化密切相关，数据库分析GalNAc-T3作用OPN蛋白O-糖基化位点。（4）液相-串联质谱技术进行蛋白质组学分析，筛选出TUSC3是14-3-3ζ介导的胆管癌细胞EMT调控糖蛋白N-糖基化的关键蛋白，KEGG通路数据库信号通路分析TUSC3调控糖蛋白N-糖基化的相关信号通路。本课题的研究发现14-3-3ζ介导胆管癌EMT转化后，通过关键分子GalNAc-T3和TUSC3分别调控OPN的O-糖基化和N-糖基化，为探寻胆管癌早期诊断标志物和靶向治疗的新靶点提供实验依据。同时，14-3-3ζ/ GalNAc-T3和14-3-3ζ/TUSC3信号通路通过介导癌细胞EMT和糖蛋白异常糖基化，使之产生“cross talk”，呈网络样调控癌细胞侵袭转移等生物学过程，可望从新的角度阐明癌细胞侵袭转移的调控机制。