Study on the mechanisms of alternative mRNA splicing that regulates the regulation of angiogenesis and diabetic complication susceptibility

在这项研究中，我们研究了选择性的前mRNA剪接/加工产生可溶性RAGE和可溶性血管内皮生长因子受体mRNAs的机制，以及它们在糖尿病血管并发症和血管生成调节中的作用。可溶性RAGE对AGE诱导的血管细胞损伤具有保护作用，而可溶性血管内皮生长因子受体是一种有效的抗血管生成因子。(1)通过RT-PCR克隆得到了海洋中相当于可溶性RAGE的基因。本研究将提供可溶性RAGE的动物同源物，以阐明其在健康和疾病中的作用。(2)我们用免疫组织化学方法研究了可溶性RAGE蛋白在人体器官和组织中的表达，发现可溶性RAGE广泛分布于各种器官和组织，包括血管内皮细胞、神经元、胰腺β细胞、巨噬细胞/单核细胞、胆管、唾液腺、消化道、肾小管、前列腺、皮肤和甲状腺。(3)我们建立了人可溶性RAGE的酶联免疫吸附试验(ELISA)，并研究了血浆可溶性RAGE水平与动脉粥样硬化的关系。并发现它与颈动脉或股动脉粥样硬化呈负相关。(4)我们利用人RAGE微基因和HEK293T细胞建立了RAGE选择性剪接的检测体系。用不同突变型RAGE微型基因对RAGE-Pre-mRNAs的顺式作用元件进行了筛选，发现了调控可溶性RAGE基因选择性剪接的顺式作用元件。我们还发现hnRNP-H参与调节可溶性RAGE mRNA的产生。(4)我们建立了利用人Flt-1微基因和原代培养的人血管内皮细胞交替3‘端处理血管内皮生长因子受体-1(Flt-1)mRNA的检测体系，并确定了Flt-1前体mRNA上的顺式作用元件，它调节了可溶性Flt-1 mRNA的产生。

In this research, we studied the mechanisms of alternative pre-mRNA splicing/processing by which mRNAs for soluble RAGE and soluble VEGF receptor are produced, and their roles in the regulation of diabetic vascular complications and angiogenesis. Soluble RAGE has a protective activity against AGE-induced vascular cell injury and soluble VEGF receptor acts as a potent anti-angiogenic factor.(1) We isolated the marine equivalent of soluble RAGE by RT-PCR cloning. This study will provide an animal orthologue of soluble RAGE to clarify its roles in health and disease.(2) We investigated the expression of soluble RAGE protein in human organs and tissues by immunohistochemical analysis, and found that soluble RAGE was widely distributed in various organs and tissues including vascular endothelium, neurons, pancreatic β cells, macrophages/monocytes, bile ducts, salivary glands, digestive tracts, renal tubules, prostate, skin, and thyroid.(3) We developed enzyme-linked immunosorbent assay (ELISA) for human soluble RAGE and examined the association of plasma soluble RAGE level with atherosclerosis, and found that it inversely correlated with carotid or femoral atherosclerosis.(4) We established an assay system for RAGE alternative splicing using a human RAGE mini-gene and HEK293T cells. Transfection experiments with various mutant RAGE mini-genes identified cis-acting elements on RAGE pre-mRNA, which regulated the alternative splicing of soluble RAGE mRNA. We also found the involvement of hnRNP-H in the regulation of soluble RAGE mRNA production.(4) We established an assay system for alternative 3'-end processing of VEGF receptor-1 (Flt-1) mRNA using a human Flt-1 mini-gene and primary cultured human vascular endothelial cells, and identified a cis-acting element on Flt-1 pre-mRNA, which regulated the production of soluble Flt-1 mRNA.