Abstract
Purpose :
ICAM-1 is a transmembrane protein that is implicated as a cancer stem-cell marker in hepatocellular cancer (HCC). The lncRNA ICR can stablilize ICAM-1 mRNA in HCC. Specific cytokines are elevated in eyes with uveal melanoma (UM). Whether ICAM-1 and ICR expression is induced by these cytokines in UM cells is unknown. We investigated whether ICR and ICAM-1 expression is altered in 4 human UM cell lines. We also investigated whether knockdown of ICR influences ICAM-1 expression levels.
Methods :
Four human UM cell lines, CRL3296, 3297, 3298 and 3299 (ATCC) were cultured and stimulated with either TNFα (10ng/ml), IL6 (100ng/ml), IL8 (100ng/ml) or DMOG (1mM). Total RNA was isolated and cDNA was prepared (N=3). Quantitative RT-PCR was used to assess fold-change (FC) in gene expression relative to untreated controls. Optimal reference genes (α-Tubulin, β-Actin, B2M, GAPDH) were determined for each cell line and each treatment. siRNA targeted to ICR and NF-κB were used to knockdown ICR and NF-κB transcripts, respectively.
Results :
Expression of the lncRNA ICR was induced by TNFα (8 - 153 FC) and IL6 (7 - 9 FC) in all UM cell lines but variation between the lines was observed. IL8 and DMOG only induced expression of ICR in one line each, 3299 (38 FC) and 3298 (23 FC), respectively. ICAM-1 expression was not induced by IL8 in any cell line, but was induced by DMOG (6 FC) in only line 3298. Except for αTubulin for TNFα and B2M for IL6 treated cell lines no single reference gene was suitable for all lines under all conditions. Increased ICR expression (38 FC) in line 3299 after IL8 treatment did not correlate with increased ICAM-1 expression, as was observed in all TNFα stimulated cells. siRNA knockdown of ICR in line 3297 resulted in downregulation of ICR (-15 FC p<0.02) and ICAM-1 (-2 FC, p<0.02). Knockdown of NF-κB message downregulated ICR (-4 FC, p<0.02) but not ICAM-1.
Conclusions :
The lncRNA ICR is expressed in UM cells and TNFα is the most potent stimulator of ICR expression for all UM cell lines tested. Genetic differences between these UM cells may influence regulation of ICR expression. Transcription factor NF-κB may regulate expression of ICR. Future experiments will determine how factors associated with UM tumor development influence ICR and ICAM-1 expression. Understanding the regulation of lncRNA expression in UM cells may open new avenues of tumor therapy.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.