Abstract 254: Apolipoprotein B Mediates Hepatic Proliferation And Cell Survival Signalling: A Crispr/cas-9 Gene Editing ApoB-knockout Study

Abstract

Introduction: Apolipoprotein B (ApoB) is a key structure protein in very-low-density lipoprotein (VLDL). Ample research has been focusing on its crucial role in lipoprotein metabolism and the onset of cardiovascular disease (CVD). However, in vivo research on the broader pathophysiological function of ApoB has been hindered by the embryonic lethality of ApoB knockout (ApoB-KO) animal models. Thus, the objective of this study is to establish ApoB-KO cell models and demonstrate ApoB as a key determinant in governing hepatocyte proliferation and apoptosis. Approaches and Results: the Crispr/Cas-9 gene editing technology was applied to genetically disrupt ApoB expression in both human and mouse hepatocytes. Total RNAs of the parental and ApoB-KO cells were subjected to RNA sequencing profiling for whole transcriptome. Subsequent bioinformatics analysis revealed that ApoB-KO significantly altered expression of more than 2000 genes in hepatocytes. More importantly, expression of genes involved in DNA replication, cell cycle, cell proliferation and apoptotic signaling were the genes mostly affected, which were either up- or down-regulated by more than 5-folds. These included the histone associated genes H2ac10-12, cyclin Ccna2, Ccnb1 and Cdc20, cell proliferation gene Fosb and apoptosis gene P53. A BrDU cell proliferation assay detecting the actively proliferating cells further demonstrated that depletion of ApoB greatly inhabited cell proliferation, indicated by the markedly reduced cell numbers and the prolonged cell doubling time in ApoB-KO cells compared to their parental cells. Mechanistic study revealed that cAMP signaling, and the insulin associated PI3K kinase pathway that medicated gene transcription, cell growth and proliferation were profoundly impaired upon ApoB depletion. Reconstitution of ApoB in the KO cells by expressing a truncated ApoB protein, ApoB72, was able to promote cell growth rate and improve cell survival to a level comparable to the parental cells. Conclusion: This is the first study that characterizes a novel function of ApoB as a key determinant in hepatic cell proliferation and survival pathways. This novel finding greatly advances our knowledge of ApoB beyond the boundary of VLDL metabolism and CVD.