程序性细胞死亡蛋白-6与扩张型心肌病的关系及其分子机制研究

Dilated cardiomyopathy (DCM), defined by ventricular chamber enlargement and systolic dysfunction, is the third major cause of heart failure, and it is also a major indication for cardiac transplantation. As little is known about the pathogenesis of DCM, to date, no early diagnosis method is available. Programmed cell death 6 (PDCD6), which was discovered in a genetic screen and proposed to have a proapoptotic function, has also been identified involved in cell proliferation, suggesting that PDCD6 may be an important modulator at the interface between cell proliferation and cell death. PDCD6 regulates the subcellular location and the JNK, a member of the MAPK pathways, activity modulation of ASK1 by direct interaction. Recently, PDCD6 has been identified as a novel p53-responsive gene and it is accumulated in the nucleus and induces apoptosis in response to DNA damage. In our previous study, two tag SNPs of PDCD6 gene, rs3756712 and rs4957014, were genotyped in 284 patients with DCM and 321 controls. Results shown that rs3756712, but not rs4957014, is associated with increased DCM risk. The PDCD6 mRNA expression level in peripheral white blood cells of DCM patients was significantly increased than that in controls. The DNA mutation in PDCD6 gene were screened by DHPLC in a small number of DCM patients and controls. A novel PDCD6 gene mutation, the C191T in mRNA resulting threonine 64 isoleucine mutation in protein (T64I), was identified in two DCM patients, while absent in controls. We constructed the PDCD6/pEGFP-C1 wild type expression plasmid, the PDCD6 C191T/pEGFP-C1 mutant expression plasmid by site-directed mutagenesis, and these plasmids were transfected into H9C2 cells. The mRNA expression levels of MCP-1, TGF-β, VEGF, SDF-1, p53 and so on were significantly influenced by the T64I mutation in PDCD6. Our previous work suggested that PDCD6 is associated with DCM, and genetic mutation(s) in PDCD6 may play an important role in the pathogenesis of DCM. In the present program, DNA mutation(s) in PDCD6 gene will be screened by DHPLC combined with DNA sequencing analysis in a larger number of samples. PDCD6 mRNA expression in peripheral white blood cells will be measured by qPCR and genetic polymorphisms will be genotyped. Mutant type of plasmid will be constructed and transfected into H9C2 cells. The influence of PDCD6 mutation(s) on the mRNA expression of MCP-1, TGF-β, VEGF, SDF-1, p53 and so on, as well as the activation of MAPK pathways (JNK, ERK, and p38) in cells cultured 24h, 48h, and 72h, will be analyzed. Our research will provide a comprehensive overview on the association between PDCD6 and DCM, and it will also clarify the hypothesis that PDCD6 mutation(s) might play an important role in the pathogenesis of DCM by influence the expression of MCP-1, TGF-β, VEGF, SDF-1, p53 and so on, and the activation of MAPK pathways. These results might contribute to the research on the pathogenesis, molecular mechanism, and early diagnosis of DCM.

扩张型心肌病(DCM)是心力衰竭的第三大病因和心脏移植的最常见病因，迄今病因不明，早期诊断十分困难。PDCD6是近年来新发现的蛋白，通过MAPK信号途径、与p53直接作用等，调控细胞凋亡/细胞增殖。课题组前期研究发现PDCD6 Tag SNP与DCM相关，DCM患者外周血白细胞中mRNA表达上调，在小样本量DCM患者中新发现T64I突变，该突变显著影响MCP-1等因子mRNA表达，强烈提示PDCD6与DCM密切相关，其突变可能是DCM的重要病因。本项目拟通过突变筛查与突变功能研究、mRNA表达及遗传多态性分析等研究，获得DCM患者中全面的PDCD6突变数据，探索突变对MCP-1等因子表达、对MAPK活化的影响。研究结果将明确PDCD6与DCM的关系，阐明突变是否通过影响MCP-1等因子表达、MAPK信号通路活化，参与DCM发病，有望为DCM病因、发病机制及早期诊断等研究提供理论与实验依据。

扩张型心肌病（DCM）是心力衰竭的第三大病因和心脏移植的最常见病因，迄今病因不明，早期诊断十分困难。PDCD6是近年来新发现的蛋白，在调控细胞凋亡/细胞增殖中发挥重要作用。本项目采用PCR-RFLP对PDCD6 Tag SNP位点进行分型，发现rs3756712的G等位基因频率在DCM组中显著小于其在对照组中频率，DCM组TG/GG基因型频率显著低于对照组；rs3756712位点TG/GG基因型与DCM患者EF、IVS及MR密切相关；生存分析结果发现rs3756712位点TG/GG基因型是DCM患者死亡的风险因素；qPCR检测外周血白细胞中mRNA表达，发现DCM患者组中PDCD6 mRNA表达水平显著增高；采用DHPLC筛查PDCD6基因突变，在DCM患者中发现T64I新突变位点；构建了pcDNA31(+)/PDCD6（WT）重组表达质粒，定点突变构建了pcDNA31(+)/PDCD6 T64I突变质粒；转染H9c2细胞后G418抗性筛选出稳转株；采用CRISPR/Cas9技术构建了PDCD6基因敲除（KO）H9c2细胞模型；CCK8检测发现：WT、T64I及KO组细胞存活率显著降低，以KO组最显著；LDH检测发现：WT、T64I及KO组细胞死亡率均显著增高，以T64I组死亡率最高；Western blot结果发现：T64I突变可能降低了PDCD6诱导AKT、p38磷酸化水平及NFκB表达的能力；分别将WT和T64I突变型mRNA显微注射斑马鱼胚胎，WISH检测myl7，发现T64I突变mRNA注射后斑马鱼胚胎中myl7基因表达显著降低。本研究结果发现：PDCD6 T64I突变可能通过影响AKT、p38磷酸化、NFκB以及myl7等基因表达，参与DCM发生发展。