Alternative Splicing in Regulation of Cholesterol Synthesis and Uptake

描述(由申请人提供)：低密度脂蛋白胆固醇水平在确定心血管疾病风险方面至关重要。最近，胆固醇生物合成的限速酶3-羟基-3-甲基戊二酰辅酶A还原酶(HMGCR)和负责摄取低密度脂蛋白的低密度脂蛋白受体(LDLR)这两个最关键的细胞内胆固醇调节因子的选择性剪接与血浆低密度脂蛋白的变化以及辛伐他汀降低低密度脂蛋白的幅度有关。最近的证据表明，与胆固醇代谢有关的四个基因(HMGCR、LDLR、HMG-CoA合成酶和甲氧戊酸激酶)的选择性剪接受到类固醇的协调调节，因此，类固醇负荷增加了选择性剪接，而类固醇耗竭抑制了选择性剪接。此外，辛伐他汀孵育的人淋巴细胞株的全基因组转录分析表明，约300个已知的转录上体成分中有95个对他汀类药物有反应(FDR&lt；0.0001)。其中，根据更多的证据，几个剪接因子参与了类固醇对选择性剪接的调节，包括：(1)基因表达的变化与细胞表面LDLR和血浆低密度脂蛋白浓度的相关性；(2)与血浆低密度脂蛋白水平相关的DNA多态；(3)siRNA下调导致前mRNA剪接的变化；以及(4)已知结合基序的电子预测。这些发现导致了一种假设，即细胞内胆固醇水平调节剪接因子(S)，以在参与胆固醇合成和吸收的多个基因的选择性剪接中产生协调变化，并且这一过程中的变化是细胞和血浆胆固醇代谢的决定因素。因此，这项提议的总体目标是：(1)证明选择性剪接是参与调节细胞胆固醇合成和摄取以及血浆低密度脂蛋白水平的一种新机制；以及(2)确定这一过程的非遗传和遗传修饰因素。为了确定在胆固醇生物合成途径中是否有更多的基因发生由类固醇调节的选择性剪接；选择性剪接的变化将在用特定的抑制剂和该途径的产物处理的HepG2细胞、原代人类肝细胞和永生化的人类淋巴细胞系中进行量化(目标1)。负责协调这些协调变化的剪接因子将使用siRNA、过度表达构建体和微型基因构建体来识别和验证(目标2)。最后，将通过测试基因调控的选择性剪接与体内血浆低密度脂蛋白水平和体外胆固醇相关表型的相关性来评估这些观察结果的生理学相关性。SNP功能将通过微基因结构的定点突变得到确认(目标3)。证明选择性剪接在调节胆固醇代谢中的作用和确定这一过程的遗传决定因素将有助于描绘导致血浆低密度脂蛋白个体间差异的分子途径，从而提高我们对心血管疾病发展和风险的理解。 与公共健康相关：这项提议的目标是测试替代剪接是否是调节胆固醇稳态的一种新机制。具体地说，我们试图确定参与胆固醇生物合成和吸收的基因在固醇调控下的选择性剪接的程度，以及识别与体内和细胞胆固醇代谢相关的这种反应的遗传和非遗传调节因子。因此，这项研究将有助于我们了解血浆低密度脂蛋白胆固醇变化的分子决定因素，进一步增加我们对心血管疾病风险和发展的了解。

DESCRIPTION (provided by applicant): LDL cholesterol levels are of fundamental importance in determining risk for cardiovascular disease. Recently, alternative splicing of the two most critical regulators of intracellular cholesterol, 3-hydroxy-3-methylglutaryl- coenzyme A reductase (HMGCR), the rate-limiting enzyme of cholesterol biosynthesis, and the LDL receptor (LDLR), responsible for uptake of LDL, have been associated with variation in plasma LDL as well as with the magnitude of LDL reduction by simvastatin. Recent evidence has indicated that alternative splicing of four genes involved in cholesterol metabolism (HMGCR, LDLR, HMG-CoA synthase and mevalonate kinase) is coordinately regulated by sterols such that sterol loading increases alternative splicing while sterol depletion suppresses alternative splicing. In addition, genome-wide transcription analysis of simvastatin incubated human lymphocyte cell lines demonstrated that 95 of ~300 known components of supraspliceosomes were responsive to statin (FDR<0.0001). Among these, several splicing factors were implicated in mediating sterol regulation of alternative splicing on the basis of additional lines of evidence including: (1) correlations of variation in gene expression with both cell surface LDLR and plasma LDL concentrations; (2) DNA polymorphisms associated with plasma LDL levels; (3) siRNA knock-down resulting in changes in pre-mRNA splicing; and (4) in silico prediction of known binding motifs. These findings lead to the hypotheses that intracellular cholesterol levels regulate splicing factor(s) to generate coordinated changes in alternative splicing of multiple genes involved in cholesterol synthesis and uptake, and that variation in this process is a determinant of cellular and plasma cholesterol metabolism. Thus, the overall objectives of this proposal are: (1) to demonstrate that alternative splicing is a novel mechanism involved in regulating cellular cholesterol synthesis and uptake as well as plasma LDL levels; and (2) to identify non-genetic and genetic modifiers of this process. To determine if sterol regulated alternative splicing occurs in a larger number of genes in the cholesterol biosynthesis pathway; changes in alternative splicing will be quantified in HepG2 cells, primary human hepatocytes, and immortalized human lymphocyte cell lines treated with specific inhibitors and products of this pathway (Aim 1). The splicing factors responsible for orchestrating these coordinated changes will be identified and validated using siRNA, overexpression constructs and mini-gene constructs (Aim 2). Lastly, the physiological relevance of these observations will be assessed by testing for associations of genetically regulated alternative splicing with both in vivo plasma LDL levels and in vitro cholesterol-related phenotypes. SNP functionality will be confirmed by site directed mutagenesis of mini-gene constructs (Aim 3). Demonstration of the role of alternative splicing in the regulation of cholesterol metabolism and identification of genetic determinants of this process will aid in delineating molecular pathways contributing to inter-individual variation in plasma LDL and thus improve our understanding of cardiovascular disease development and risk. PUBLIC HEALTH RELEVANCE: The goal of this proposal is to test if alternative splicing is a novel mechanism of regulating cholesterol homeostasis. Specifically, we seek to determine the extent of sterol regulated alternative splicing of genes involved in cholesterol biosynthesis and uptake, as well as to identify both genetic and non-genetic regulators of this response that are associated with both in vivo and cellular cholesterol metabolism. Thus, this research will contribute to our understanding of molecular determinants of variation in plasma LDL cholesterol, further adding to our knowledge of cardiovascular disease risk and development.