Tumor-specific expression and alternative splicing of the COL6A3 gene in pancreatic cancer.
Tumor-specific expression and alternative splicing of the COL6A3 gene in pancreatic cancer.
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DOI：
10.1016/j.surg.2011.05.011
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发表时间：
2011-08
期刊：
影响因子：
3.8
通讯作者：
Chu ML
中科院分区：
文献类型：
--
作者：
Arafat H;Lazar M;Salem K;Chipitsyna G;Gong Q;Pan TC;Zhang RZ;Yeo CJ;Chu ML
胰腺导管腺癌(PDA)是一种高度致命的疾病，其显著的促结缔组织增生性反应是其特征。纤维性胶原，如I型胶原和较小程度的III型和V型胶原构成了间质纤维化的大部分。VI型胶原(COL6)形成与I型胶原纤维相关的微纤维网络。COL6的表达与炎症和生存有关。重要的是，基因组外显子阵列已经在几种癌症中发现了COL6A3中肿瘤特异性的选择性剪接。我们评估了COL6A3在PDA和癌前病变中的表达和定位，并探索了不同剪接事件的存在。我们用RT-PCR技术分析了成对的PDA-Normal(n=18)、IPMN(n=5)、胰腺囊腺瘤(n=5)和8个PDA细胞系，用独特的引物鉴定了COL6A3基因及其几个外显子上的选择性剪接位点。采用Western印迹分析和免疫组织化学方法分析COL6A3蛋白在不同皮损和两种PDA动物模型中的表达水平和定位。在所检测的配对PDA邻近组织中，有77%的COL6A3蛋白水平显著上调。COL6A3主要表达于PDA的促结缔组织间质，在恶性导管周围和间质脂肪浸润处有较高的沉积。对COL6A3剪接变异体的分析显示，在18例(94%)成对的PDA邻近组织中，有17例(94%)肿瘤特异的第3和第6外显子的包涵体。外显子4的包含完全是肿瘤特异性的，在邻近组织中几乎检测不到表达。IPMN和胰腺囊腺瘤均未检测到任何外显子的表达。在6个PDA细胞系中检测到总的COL6A3mRNA和外显子6，但只有两个细胞系(MIA PACA-2和ASPC-1)表达外显子3和4。在PDA的异种移植和转基因模型中，COL6A3在间质和部分PDA细胞中均有表达。我们首次描述了肿瘤特异性选择性剪接在间质COL6A3的几个外显子中的动态过程。或者，剪接蛋白可能有助于癌症的病因或进展，并可能作为癌症诊断的标记物。将COL6A3亚型鉴定为PDA特异的亚型，为进一步研究这些选择性剪接事件的致癌和诊断潜力提供了基础。
Pancreatic ductal adenocarcinoma (PDA) is a highly lethal disease in which a prominent desmoplastic reaction is a defining characteristic. Fibrillar collagens, such as collagen I and to a lesser extent, collagen III and V comprise the majority of this stromal fibrosis. Type VI collagen (COL6) forms a microfibrillar network associated with type I collagen fibrils. The expression of COL6 has been linked to inflammation and survival. Importantly, tumor-specific alternative splicing in COL6A3 has been identified in several cancers by genome exon arrays. We evaluated the expression and localization of COL6A3 in PDA and premalignant lesions and explored the presence of alternative splicing events. We analyzed paired PDA-normal (n=18), IPMN (n=5), pancreatic cystadenoma (n=5), and eight PDA cell lines with RT-PCR, using unique primers that identify total COL6A3 gene and alternative splicing sites in several of its exons. Western blot analysis and immunohistochemistry were used to analyze the expression levels and localization of COL6A3 protein in the different lesions, and in two animal models of PDA. COL6A3 protein levels were significantly upregulated in 77% of the paired PDA-adjacent tissue examined. COL6A3 was mainly present in the desmoplastic stroma of PDA, with high deposition around the malignant ducts and in between the sites of stromal fatty infiltration. Analysis of the COL6A3 splice variants showed tumor-specific consistent inclusion of exons 3 and 6 in 17 of the 18 (94%) paired PDA-adjacent tissues. Inclusion of exon 4 was exclusively tumor-specific, with barely detectable expression in the adjacent tissues. IPMN and pancreatic cystadenomas showed no expression of any of the examined exons. Total COL6A3 mRNA and exon 6 were identified in six PDA cell lines, but only two cell lines (MIA PACA-2 and ASPC-1) expressed exons 3 and 4. In both the xenograft and transgenic models of PDA, COL6A3 immunoreactivity was present in the stroma and some PDA cells. We describe, for the first time, a dynamic process of tumor-specific alternative splicing in several exons of stromal COL6A3. Alternatively spliced proteins may contribute to the etiology or progression of cancer and may serve as markers for cancer diagnosis. Identification of COL6A3 isoforms as PDA-specific provides the basis for future studies to explore the oncogenic and diagnostic potential of these alternative splicing events.
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