胰腺癌小富亮氨酸重复蛋白糖胺聚糖硫酸酯化模式的研究

The small leucine-rich repeat proteoglycans (SLRPs) are important components of the extracellular matrix (ECM) that bind transforming growth factor-beta (TGF-β) and other matrix molecules, thereby regulating cellular proliferation, angiogenesis and immune responses. Post-translational modifications of proteoglycans (PGs) including keratocan, lumican, mimecan and decorin, attachment of sulfate groups to their glycosaminoglycan (GAG) chains, add a level of coding information that contributes greatly to specific interactions of these proteins with other molecules. The PGs in pancreatic cancer are produced by at least two cell types, including fibroblasts of the tumor stroma and pancreatic carcinoma cells. We propose the following hypothesis: SLRPs produced by pancreatic tissue have specific sulfation characteristics. SLRPs play essential roles in the regulation of pancreatic carcinoma growth and angiogenesis. The mechanism of this regulation involves changes in sulfation characteristics of their GAG chains. To test this hypothesis we will perform a series of experiments aimed at: (i) Identifying the SLRPs that are expressed in pancreatic cancer (ii) Characterizing the sulfation patterns of their GAG chains, (iii) Assessing the interactions between SLRPs/GAGs and other ECM molecules such as TGF-β, collagens and matrix metalloproteinases (MMPs), etc. A combination of immunoblotting, enzyme digestions for glycan analysis and electrospray ionization tandem mass spectrometry (ESI-MS/MS), and surface plasmon resonance (SPR) techniques will be used in these experiments. Significance: The types and degrees of glycosylation and sulfation of SLRPs in pancreatic cancer are not known. Similarly, molecular mechanisms of growth and metastasis of pancreatic carcinoma are poorly understood. The proposed research may have an immediate impact on understanding the biology of pancreatic cancer. Moreover, our studies will pioneer a new approach that will directly test and emphasize the role of post translational modifications of SLRPs in the biology of cancer and may offer potential new therapeutic targets for the treatment of this disease.

小富含亮氨酸重复蛋白聚糖（SLRPs）是细胞外基质的重要组成部分。蛋白聚糖如keratocan，lumican，mimecan和decorin等的翻译后修饰，特别是硫酸基团结合糖胺聚糖链(GAGs)上即硫酸酯化，将有助于与其他分子特异性作用。我们提出以下假设：胰腺癌产生的SLRPs的GAGs含有特定的硫酸基序列，该硫酸基位点序列对SLRPs与其他基质分子的相互结合起关键作用。为验证这一假设，本项目采用免疫亲和层析、糖苷酶水解、液质串联质谱、表面等离子体共振技术进行一系列试验，旨在系统研究胰腺癌组织及胰腺癌细胞类群表达的SLRPs特征；阐明SLRPs糖链的硫酸基团序列；评价SLRP及其GAGs和其他基质分子的相互作用。本研究有助于理解胰腺癌生长、发育及转移的分子机制，可望为胰腺癌早期诊断提供新的标志物，为临床治疗提供潜在的新靶点。

胰腺癌 (PC) 是癌症相关死亡率的第七大原因，并且在所有癌症类型中表现出最低的五年生存率 (9%)。细胞外基质 (ECM) 是向肿瘤细胞提供生物物理和生化信号的主要成分。小富含亮氨酸重复蛋白聚糖（SLRPs）如keratocan，lumican，mimecan和Decorin是细胞外基质的重要组成部分。本课题对胰腺癌组织及胰腺癌细胞类群表达的keratocan，lumican，mimecan和Decorin糖胺聚糖硫酸酯化模式及其核心蛋白调控胰腺癌生长、发育的分子机制进行了探索。结果表明：ΔDi-4S，ΔDi-6S，KS-6S和KS-6S、6'S在胰腺癌中的含量显著升高。糖胺聚糖CS/DS硫酸化糖基转移酶CHST3、CHST12、CHST13 和 CHST15与胰腺癌的增殖和转移直接相关。CHST3、CHST12、CHST13 和 CHST15 表达谱对胰腺肿瘤的早期临床诊断提供了潜在的生物标志物。证实了胰腺癌中Decorin表达量升高主要是由于其剪接体Decorin B、C和D的升高导致，其与完整核心蛋白Decorin A不同，Decorin核心蛋白聚糖 B能够促进癌症的增殖和迁移，抑制细胞凋亡。Decorin核心蛋白聚糖 B 在 PC 的进展中发挥了至关重要的作用。我们发现Lumican在PDA癌组织中表达水平高于癌旁胰腺组织，p21、p53在PDA癌组织中表达水平低于癌旁胰腺组织。干扰BXPC-3细胞lumican的表达能够增强细胞活力，并抑制细胞凋亡。同时发现胰腺癌中keratocan在PDA癌组织中表达水平高于癌旁胰腺组织，keratocan过表达可增强BXPC-3细胞活力，并通过调控 p21、 p53蛋白的表达促进胰腺癌的发生发展。另外，我们发现Osteoglycin（OGN）在 PC 组织中显着上调，高水平 OGN 的 PC 患者存活率低。 OGN过表达促进了PC细胞的增殖、迁移和侵袭，抑制了PC细胞的凋亡。OGN 过表达上调了 DNA 结合 4 (ID4) ， ID4 介导 OGN 对 PC 细胞的影响。此次研究发现，OGN基因在胰腺癌的发生发展中发挥重要作用。在今后的临床胰腺癌诊疗中，OGN基因有望成为有效的血清标志物。同时，调节OGN的表达水平或可成为治疗胰腺癌的有效方法。本研究结果有助于理解胰腺癌生长、发育及转移的分子机制。