Novel genetic regulators of fibrinogen synthesis identified by an in vitro experimental platform

Background: Fibrinogen has an established, essential role in both coagulation and in-flammatory pathways, and these processes are deeply intertwined in the development of thrombotic and atherosclerotic diseases. Previous studies aimed to better under-stand the (patho) physiological actions of fibrinogen by characterizing the genomic contribution to circulating fibrinogen levels.Objectives: Establish an in vitro approach to define functional roles between genes within these loci and fibrinogen synthesis. Methods: 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.Results: HepG2 cells secreted fibrinogen, and stimulation with interleukin-6 increased fibrinogen production by 3.4 & PLUSMN; 1.2 fold. In the absence of interleukin-6, small inter-fering 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 fibrin-ogen 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.Conclusions: 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.

Automated summary

This study aimed to understand the (patho) physiological actions of fibrinogen by characterizing the genetic contribution to its synthesis. By inducing HepG2 cells to express fibrinogen and observing the effects of small interfering RNA knockdown of different candidate genes, several genes interacting with fibrinogen production were identified. This approach can reveal cellular mechanisms of fibrinogen and fibrin(ogen)-mediated (patho) physiological mechanisms.