Silencing COQ8B in Aortic Smooth Muscle Cells Reveals Cellular Dysfunction Related to Changes in Cell Proliferation

Abstract

Background/Objective: Thoracic aortic aneu­rysm (TAA) is a prevalent disorder that predis­poses to aortic dissection. Prior work identified the ubiquinone biosynthesis gene COQ8B as a genetic modifier of TAA progression. We sought to determine the impact of decreased COQ8B on global transcription in aortic smooth muscle cells (SMCs).

Methods: Primary human aortic SMCs from a healthy donor were seeded in 12-well plates. Six experimental conditions were created, each with 3 replicates: 1) siRNA targeting COQ8B (siCO­Q8B); 2) siRNA targeting the dominant TAA gene SMAD3 (siSMAD3); 3) negative control siRNA (siNeg); 4) siCOQ8B and siSMAD3; 5) siCOQ8B with Angiotensin II (AngII) stimula­tion (siCOQ8B+AngII); 6) siNeg+AngII. RNA was extracted approximately 48 hours post-siR­NA transfection and, for AngII conditions, after 1 hour of incubation with AngII (100 nM). mRNA-sequencing was performed and down­stream analysis utilized R packages EdgeR and topGO.

Results: Multidimensional scaling identified dis­tinct clustering of samples by experimental con­dition. Downregulated genes in siCOQ8B were enriched for Gene Ontology pathways related to cell proliferation including cell cycle regulators, DNA replication, and mitosis. MYOCD, a mas­ter regulator of SMC homeostasis, was down­regulated. Similar proliferation-related pathways were enriched in siCOQ8B+siSMAD3 and siCOQ8B+AngII compared to siNeg. Pathways related to cell proliferation in siCOQ8B+AngII cells were downregulated when compared to siCOQ8B which indicates that AngII infusion in the context of COQ8B silencing may further dysregulate cell proliferation pathways.

Conclusion/Implications: The results indi­cate that COQ8B has an important role in cell cycle processes in aortic SMCs, including when SMCs are exposed to stressors associated with TAA development. Stimulation of angiotensin receptors may exacerbate the effects of decreased COQ8B in these processes. To investigate these experimental results in human pathology, bulk RNA samples and intact nuclei have been iso­lated from frozen human aortic specimens and prepared for transcriptomic analysis.