Department of Pathology and Laboratory Medicine and UNC Blood Research Center, University of North Carolina at Chapel Hill, NC, USA.
Department of Biostatistics and North Carolina Translational and Clinical Sciences Institute, University of North Carolina at Chapel Hill, NC, USA.
J Thromb Haemost. 2023 Mar;21(3):522-533. doi: 10.1016/j.jtha.2022.10.027. Epub 2022 Dec 22.
Fibrinogen has an established, essential role in both coagulation and inflammatory pathways, and these processes are deeply intertwined in the development of thrombotic and atherosclerotic diseases. Previous studies aimed to better understand the (patho) physiological actions of fibrinogen by characterizing the genomic contribution to circulating fibrinogen levels.
Establish an in vitro approach to define functional roles between genes within these loci and fibrinogen synthesis.
Candidate genes were selected on the basis of their proximity to genetic variants associated with fibrinogen levels and expression in hepatocytes and HepG2 cells. HepG2 cells were transfected with small interfering RNAs targeting candidate genes and cultured in the absence or presence of the proinflammatory cytokine interleukin-6. Effects on fibrinogen protein production, gene expression, and cell growth were assessed by immunoblotting, real-time polymerase chain reaction, and cell counts, respectively.
HepG2 cells secreted fibrinogen, and stimulation with interleukin-6 increased fibrinogen production by 3.4 ± 1.2 fold. In the absence of interleukin-6, small interfering RNA knockdown of FGA, IL6R, or EEPD1 decreased fibrinogen production, and knockdown of LEPR, PDIA5, PLEC, SHANK3, or CPS1 increased production. In the presence of interleukin-6, knockdown of FGA, IL6R, or ATXN2L decreased fibrinogen production. Knockdown of FGA, IL6R, EEPD1, LEPR, PDIA5, PLEC, or CPS1 altered transcription of one or more fibrinogen genes. Knocking down ATXN2L suppressed inducible but not basal fibrinogen production via a post-transcriptional mechanism.
We established an in vitro platform to define the impact of select gene products on fibrinogen production. Genes identified in our screen may reveal cellular mechanisms that drive fibrinogen production as well as fibrin(ogen)-mediated (patho)physiological mechanisms.
纤维蛋白原在凝血和炎症途径中都具有既定的、必不可少的作用，这两个过程在血栓形成和动脉粥样硬化疾病的发展中深深地交织在一起。先前的研究旨在通过描述对循环纤维蛋白原水平的遗传贡献来更好地了解纤维蛋白原的（病理）生理作用。
建立一种体外方法来确定这些基因座内的基因与纤维蛋白原合成之间的功能关系。
根据它们与纤维蛋白原水平相关的遗传变异以及在肝细胞和 HepG2 细胞中的表达，选择候选基因。用靶向候选基因的小干扰 RNA 转染 HepG2 细胞，并在不存在或存在促炎细胞因子白细胞介素-6 的情况下培养。通过免疫印迹、实时聚合酶链反应和细胞计数分别评估纤维蛋白原蛋白产生、基因表达和细胞生长的影响。
HepG2 细胞分泌纤维蛋白原，白细胞介素-6 刺激使纤维蛋白原的产生增加了 3.4±1.2 倍。在没有白细胞介素-6 的情况下，FGA、IL6R 或 EEPD1 的小干扰 RNA 敲低降低了纤维蛋白原的产生，而 LEPR、PDIA5、PLEC、SHANK3 或 CPS1 的敲低增加了其产生。在有白细胞介素-6 的情况下，FGA、IL6R 或 ATXN2L 的敲低降低了纤维蛋白原的产生。FGA、IL6R、EEPD1、LEPR、PDIA5、PLEC 或 CPS1 的敲低改变了一个或多个纤维蛋白原基因的转录。敲低 ATXN2L 通过转录后机制抑制了诱导但不抑制基础纤维蛋白原的产生。
我们建立了一个体外平台来确定选定基因产物对纤维蛋白原产生的影响。我们筛选出的基因可能揭示了驱动纤维蛋白原产生的细胞机制以及纤维蛋白原介导的（病理）生理机制。
