miR-608 rs4919510C/G 影响严重创伤患者预后的机制研究

miRNAs are post-transcriptional regulators that bind to complementary sequences on target messenger RNA transcripts (mRNAs), usually resulting in translational repression or target degradation and gene silencing. They have been demonstrated to play important role in inflammation and immune response. Our previous studies have found out that miR-608 rs4919510 C/G has close relationship with the morbidity of complications, such as sepsis and multiple organ failure, in severe trauma patients through multi-center clinical trials. rs4919510G has also been found to be associated with higher levels of mature miR-608, suggesting that it maybe a functional single nucleotide polymorphism. In order to further clarify the functional significance of rs4919510, we attempted to found out the effect of rs4919510 on the mature processor of miR-608 and miR-608's interaction with its target genes, using quantitative PCR, magnetic bead screening, expression microarray and other in vitro experiments. We also wanted to clarify miR-608's target genes in THP-1 or U937 cells. Furthermore, we reconfirm our results using samples from trauma patients with different genotypes. This study was designed to demonstrate the functional significance of miR-608 rs4919510 and clarify the mechanism of its impact on the outcome of the severe trauma patients. It will provide a new molecular marker for the early warning of complications of severe trauma patients.

miRNA是基因转录后水平调节的重要分子，与炎症、免疫反应密切相关。前期研究通过多中心临床实验，系统筛选与严重创伤并发症相关的miRNA基因多态性，发现miR-608 rs4919510C/G在多个人群中与脓毒症、多器官功能衰竭发病率密切相关，且能提高细胞内成熟miR-608表达水平，提示其可能为一个功能性SNP位点。为深入研究该位点的功能意义，本课题拟从其影响miR-608成熟和miR-608与靶基因相互作用两方面，利用定量PCR、磁珠筛选和表达谱芯片等系列体外实验，阐明其影响miR-608成熟的具体机制，明确miR-608在THP-1/U937细胞中的靶基因，证实rs4919510在其中的作用；进一步利用不同基因型的严重创伤患者标本进行在体验证。研究旨在从功能水平上明确miR-608 rs4919510影响严重创伤患者预后的机制，为严重创伤并发症的预警诊断提供新的分子标志。

MiR-608是近年来新发现的miRNA。我们及其他研究提示其在脓毒症、肿瘤等多种炎症相关疾病的发病机制中发挥重要作用。miR-608 rs4919510C/G位点被发现与脓毒症、多器官功能衰竭发病率密切相关，可能为功能性多态性位点。为明确miR-608及其多态性位点的功能意义，我们首先利用多中心大样本，进一步验证了miR-608 rs4919510位点与创伤并发症发生率之间的相关性，发现在重庆（n=666），云南（n=286）及浙江（n=316）人群中rs4919510分别与脓毒症的发病率和/或MODS评分升高相关，且在重庆（P=0.034）、浙江（P=0.012）和合并人群中（P=1.13×10-5）差异显著。生物信息学分析显示rs4919510变异可引起其发夹结构自由能升高（-30.30kcal/mol vs. -31.10kcal/mol）和碱基配对结构的改变。体外转染实验也证实突变型质粒转染后细胞内成熟miR-608的表达水平显著高于野生型质粒（P=0.002）。为进一步明确miR-608在单核细胞中的靶基因，我们通过慢病毒转染技术，建立了miR-608低表达THP-1细胞系。通过表达谱芯片检测方法筛选出miR-608在THP-1细胞中可能的靶基因。运用qPCR、Westernblot等体外实验，我们证实干扰细胞内成熟miR-608水平可以显著影响ELANE基因的mRNA和蛋白表达水平，提示ELANE基因可能为miR-608在单核细胞中的靶基因。生物信息学分析提示miR-608与ELANE基因结合的位点很可能在其外显子区域内。该结果通过报告基因实验进一步证实。研究结果提示miR-608是ELANE基因在单核细胞中表达的重要调节分子。miR-608及其靶基因ELANE很可能成为脓毒症新的生物标志物或治疗靶点。