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具有重要意义。最近，细胞内胆固醇的两个最关键的调节因子，3-羟基-3-甲基戊二酰辅酶A还原酶（HMGCR），胆固醇生物合成的限速酶，和LDL受体（LDLR），负责LDL的摄取，剪接的选择与血浆LDL的变化以及辛伐他汀降低LDL的幅度有关。最近的证据表明，参与胆固醇代谢的四个基因（HMGCR、LDLR、HMG-CoA合成酶和甲羟酸激酶）的选择性剪接受到甾醇的协调调节，因此甾醇负载增加了选择性剪接，而甾醇耗尽抑制了选择性剪接。此外，对辛伐他汀培养的人淋巴细胞系的全基因组转录分析表明，在约300种已知的上质体成分中，有95种对他汀类药物有反应（FDR<0.0001）。其中，一些剪接因子与介导选择性剪接的固醇调节有关，这些证据包括：(1)基因表达变异与细胞表面LDLR和血浆LDL浓度的相关性；(2)与血浆LDL水平相关的DNA多态性；(3) siRNA敲除导致pre-mRNA剪接改变；(4)已知结合基序的计算机预测。这些发现导致了这样的假设：细胞内胆固醇水平调节剪接因子，从而在参与胆固醇合成和摄取的多个基因的选择性剪接中产生协调的变化，而这一过程的变化是细胞和血浆胆固醇代谢的决定因素。因此，本研究的总体目标是：(1)证明选择性剪接是一种参与调节细胞胆固醇合成和摄取以及血浆LDL水平的新机制；(2)鉴定该过程的非遗传修饰因子和遗传修饰因子。确定胆固醇生物合成途径中是否有更多的基因存在由甾醇调节的选择性剪接；在HepG2细胞、原代人肝细胞和永生的人淋巴细胞系中，用该途径的特异性抑制剂和产物处理后，将量化选择性剪接的变化（目的1）。负责协调这些协调变化的剪接因子将使用siRNA、过表达构建体和迷你基因构建体进行鉴定和验证（目的2）。最后，这些观察结果的生理相关性将通过检测体内血浆LDL水平和体外胆固醇相关表型与遗传调控的选择性剪接的关联来评估。SNP的功能将通过位点定向诱变对微型基因结构进行确认（目的3）。证明选择性剪接在胆固醇代谢调节中的作用以及确定这一过程的遗传决定因素将有助于描述导致血浆LDL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。

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.