Expression Profiling of Long Noncoding RNA Splice Variants in Human Microvascular Endothelial Cells: Lipopolysaccharide Effects In Vitro

Abstract

Endothelial cell interactions with lipopolysaccharide (LPS) involve both activating and repressing signals resulting in pronounced alterations in their transcriptome and proteome. Noncoding RNAs are now appreciated as posttranscriptional and translational regulators of cellular signaling and responses, but their expression status and roles during endothelial interactions with LPS are not well understood. We report on the expression profile of long noncoding (lnc) RNAs of human microvascular endothelial cells in response to LPS. We have identified a total of 10,781 and 8310 lncRNA transcripts displaying either positive or negative regulation of expression, respectively, at 3 and 24 h posttreatment. A majority of LPS-induced lncRNAs are multiexonic and distributed across the genome as evidenced by their presence on all chromosomes. Present among these are a total of 44 lncRNAs with known regulatory functions, of which 41 multiexonic lncRNAs have multiple splice variants. We have further validated splice variant-specific expression of EGO (NONHSAT087634) and HOTAIRM1 (NONHSAT119666) at 3 h and significant upregulation of lnc-IL7R at 24 h. This study illustrates the genome-wide regulation of endothelial lncRNA splice variants in response to LPS and provides a foundation for further investigations of differentially expressed lncRNA transcripts in endothelial responses to LPS and pathophysiology of sepsis/septic shock.

Figure 1

Schematic of (a) NONHSAT087634 and (b) NONHSAT119666 splice variant specific primer selection.

Figure 2

Schematic illustration of the procedure for identification, cataloging, and validation of lncRNA transcripts.

Figure 3

Chromosome-wise distribution of differentially expressed lncRNA transcripts in HMECs after LPS treatment. (a) Upregulated transcripts after 3 h. (b) Downregulated transcripts after 3 h. (c) Upregulated transcripts after 24 h. (d) Downregulated transcripts after 24 h.

Figure 4

Cataloging of differentially expressed lncRNA transcripts from LPS-treated HMECs based on (a) strand-specific origin, (b) lengthwise distribution, and (c) number of exons.

Figure 5

Identification of functionally annotated lncRNAs in HMECs treated with LPS for 3 and 24 h. Differentially expressed lncRNAs belong to different categories based on publicly available databases and published reports.

Figure 6

Bar diagram represents total splice variants available for 44 functionally annotated lncRNAs and their respective differentially expressed splice variants in HMECs after LPS treatment.

Figure 7

Quantitative RT-PCR for EGO (NONHSAT087634), HOTAIRM1 (NONHSAT119666), and lnc-IL7R expression in HMECs following LPS treatment for (a) 3 h and (b) 24 h (^∗P ≤ 0.05 and ^∗∗∗P ≤ 0.001; and ns = nonsignificant).