LRP6基因调控区变异调控血管内皮细胞Wnt通路的机制研究

Coronary artery disease is one of the most common cardiovascular diseases. In China, the mortality of CAD has reached second in the world, and more than 700 thousand people die every year from coronary artery disease. It has been reported that mutations in the protein coding region of the low density lipoprotein receptor related protein 6 (LRP6) are associated with familial coronary artery disease, which can lead to inadequate activation of the Wnt signaling pathway. However, in patients with late-onset CAD, variants in the coding region of LRP6 gene was not found to be associated with the disease, one SNP in the second intron was associated with the susceptibility of CAD. In our previous research, a SNP rs60730865, located in the promoter of LRP6, was found to be linked with CAD. The SNP is located in the DNA binding motif of ATF5 (activating transcription factor 5). It’s speculated that this SNP can affect the binding capacity of ATF5, thereby regulating the expression of LRP6. Therefore, we put forward a hypothesis that LRP6 can regulate the activation of Wnt pathway not only by missense mutations in the coding region, but also by regulatory variants in the promoter region to affect the expression level of LRP6. The hypothesis will be verified through different assays, such as luciferase reporter assay, EMSA assay and chromatin immunoprecipitation assay to study the mechanisms of the newly discovered CAD-associated variant for regulating LRP6 gene expression. This study will broaden our horizon about the genetic factors of CAD from a new perspective.

冠状动脉疾病是一种最常见的心血管病。在中国，CAD死亡人数已高居世界第二位，每年有超过70万人死于冠状动脉疾病。有研究发现LRP6基因的蛋白编码区突变与家族性冠状动脉疾病有关，这些突变可导致Wnt信号通路活化水平不足。然而在迟发性CAD中，LRP6蛋白编码区并没有发现与疾病相关的突变，却在其第二内含子中发现了与疾病易感性相关的位点。在前期研究，位于LRP6启动子区的rs60730865与CAD有关，该SNP位于ATF5的DNA结合元件内，可影响ATF5的结合能力，进而调节LRP6的表达水平。因此我们认为，LRP6除了通过编码区突变调节Wnt通路活化水平外，还可能通过改变LRP6的表达水平进而影响Wnt通路的活化程度，参与疾病的发生过程。本项目拟通过荧光素酶报告基因实验、EMSA实验、染色质免疫共沉淀实验等来研究新发现的变异位点调节LRP6基因表达的分子机制，从新的角度了解CAD的遗传因素。

本研究收集500例早发CAD患者样本及500例健康对照，对LRP6 基因中的SNP （rs17848270，rs201730937，rs7978064，rs2302686，rs2302685，rs1012672）进行分型，发现rs17848270位点与早发CAD患者存在显著的关联（P<0.0001）； 本课题组对40例冠心病（CAD）患者和40例健康体检者进行16种白细胞亚群的分布与计数实验，发现冠心病患者所有单核细胞、未成熟粒细胞和B淋巴细胞的百分比和计数显著增加，表明这些白细胞亚群的水平可能是诊断冠心病的潜在生物标志物。CAD患者的细胞毒性T/自然杀伤淋巴细胞水平明显降低；本课题组探索小而密低密度脂蛋白胆固醇（sdLDL-C）及其与低密度脂蛋白胆固醇之比（sdLDL-C /LDL-C）与颈动脉粥样硬化斑块的关系，发现sdLDL-C和sdLDL-C /LDL-C的水平在斑块组和IMT增厚组明显高于对照组。在LDL-C水平正常的受试者中(LDL-C <2.59 mmol/L)，颈动脉内膜异常组的sdLDL-C和sdLDL-C/LDL-C比值水平依然高于对照组。sdLDL-C是颈动脉IMT增厚和斑块的独立危险因素；本研究对60例DCM心力衰竭患者、52例CAD性心力衰竭患者和70例健康体检者检测进行可溶性ST2与半乳凝集素3，发现DCM组与健康对照组比较，血清sST2水平显著升高，血清 gal-3水平也显著增高。DCM组与CHD组相比，血清gal-3水平明显升高；本研究对10例早发CAD患者和10例健康者进行转录组测序，发现FN1、F5、GATA4和FADS1这4个基因在CAD患者中表达上调，HSPA1A基因表达下调，此结果表明GATA4基因很可能是早发CAD患者中的特异基因。