RNA-sequencing of human vascular endothelial cells after si-RNA mediated gene silencing of interleukin-6 (IL6)

Purpose: To characterize the autocrine functions of IL6 in human endothelial cells Methods: Knockdown of interleukin-6 in cultured human umbilical vein endothelial cells were performed using stealth-siRNA and lipofectamine 2000. Total RNA was isolated 48h after siRNA introduction. RNA sequencing libraries were prepared using TruSeq stranded mRNA sample prep kit including poly-A selection. Sequencing was performed on a Illumina HiSeq2500 instrument. Results: Knockdown of IL6 in human endothelial cells resulted in 84-92% reduction in the release of IL6. Knockdown of IL6 in the absence of soluble IL6Ra (sIL6Ra) lead to differential regulation of 1915 genes (using a fold change 1.5, q < 0.05), and 1967 differentially regulated genes in the presence of sIL6Ra. Treatment with sIL6Ra alone had a limited effect and resulted only in 59 differentially expressed genes. Conclusion: Knockdown of IL6 in human endothelial cells results in dramatic changes in transcriptional patterns, which seem to be independent of sIL6Ra, indicating that the autocrine functions of IL6 in human endothelial cells are mainly mediated via classic IL6 signaling. Overall design: siRNA mediated gene silencing of interleukin-6 followed by RNA sequencing was performed in cultured human endothelial cells.