PRSS3介导的CD109裂解位点鉴定及其裂解片段在胰腺癌中的作用

Metastasis accounts for the poor outcome of patients with pancreatic cancer. we previously found that PRSS3 upregulates VEGF expression via the PAR1-mediated ERK pathway in human pancreatic cancer, but no significant correlation between VEGF expression and the growth and invasion of pancreatic cancer cells was observed.Recently, we found that CD109 expression was upregulated by PRSS3 and then was processed into 120kDa and 70kDa CD109 by PRSS3 cleavage. The expression and cleavage of CD109 protein enhanced the malignant growth mediated by PRSS3 in pancreatic cancer. However, the PRSS3 cleavage site and the potential significance of 120kDa and 70kDa CD109 fragments are not yet understood. In this study, we will identify the PRSS3 cleavage site on CD109 protein and examine the mechanism of action of 120kDa and 70kDa CD109. Meanwhile, the amount of CD109 fragments will be detected by ELISA from the sera of patients with pancreatic cancer. The statistical analysis between the expression of PRSS3 and CD109 will be performed in combination with clinical pathological data. This project will elucidate the function of 120kDa and 70kDa CD109 cleavaged by PRSS3 and their effect on growth, progression and metastasis of pancreatic cancer. The cleavage site of CD109 by PRSS3 may represent a novel therapeutic target for treatment of this disease.

复发与转移是导致胰腺癌预后差的主要原因。我们前期研究结果显示三型胰酶原PRSS3能促进胰腺癌转移和降低生存率。最近我们发现PRSS3能够诱导胰腺癌细胞CD109的表达并在细胞外将CD109裂解成120kDa和70kDa两个片段，CD109的诱导表达及其裂解增强了PRSS3作用在肿瘤细胞上的恶性表型。本研究拟应用肽谱分析技术鉴定胰腺癌细胞CD109被PRSS3裂解的位点，然后纯化活性裂解片段，结合体内外实验及分子生物学技术研究其作用机制。免疫组化方法检测胰腺癌患者组织PRSS3和CD109表达相关性，结合ELISA法检测患者血液中CD109裂解活性片段的含量，经过统计分析探讨胰腺癌中PRSS3诱导CD109表达极其裂解作用对转移和预后的影响。本课题的实施可望阐明胰腺癌恶性进展的原因和为胰腺癌靶向治疗提供理论依据。

项目的背景：胰腺癌因其发病隐匿而成为恶性程度最高的肿瘤之一。我们以往研究证实PRSS3的高表达与胰腺癌生存呈负相关，分泌到细胞外的PRSS3裂解细胞膜蛋白CD109。本项目分析裂解位点及研究裂解产物在胰腺癌中的作用机制及临床应用价值。.研究内容：用Western blotting检测8个人胰腺癌细胞系CD109蛋白表达水平；生物素标记胰腺癌细胞Patu8988s与活性PRSS3孵育后，收集上清中脱落的tCD109片段，用nanoLC-MS / MS法进行N端氨基酸序列分析；根据CD109的裂解位点构建tCD109分泌型表达载体pSec-tCD109；将 pSec-tCD109转染到Patu8988t-tCD109或Miapac2-tCD109细胞，观察tCD109对肿瘤细胞增值、迁移和浸润的影响；应用免疫共沉淀和表面等离子共振技术（SPR）研究tCD109与TGF-β受体的作用关系及其调节细胞生物学行为的信号通路；收集58例胰腺癌患者血清标本，用ELISA方法检测血浆中tCD109含量，并分析tCD109的含量与胰腺癌患者生存关系。.重要结果：在胰腺癌细胞中，PRSS3的表达能够裂解细胞膜表面CD109蛋白，裂解位点在R663-F664之间；蛋白裂解片段V22-R663即tCD109可促进胰腺癌细胞系增值侵袭和转移能力； tCD109与TGF-β1竞争性的结合TGF-β受体,从而抑制其下游信号传导；胰腺癌患者血清中tCD109检测可用于早期诊断及判断患者预后。.关键数据：PRSS3裂解CD109位点在R663-F664之间；血清中tCD109检测方法用于胰腺癌诊断的特异性达到100%，而敏感性65.5%。.科学意义：选取胰腺癌为研究对象，分析了PRSS3裂解CD109的产物对肿瘤的作用机制，建立了胰腺癌早期诊断及预后判断的检测方法，同时为胰腺癌靶向治疗奠定了重要的分子理论基础。