C. L. Bishop1, S. O’Brien1, L. Dailey1, V. Stephen1, M. Eichenberger1, S. Galandiuk1 1University of Louisville School of Medicine,Price Institute of Surgical Research,Louisville, KY, USA
Introduction: Colorectal cancer (CRC) is the fourth leading cause of cancer-related deaths in the U.S. Long non-coding RNAs (lncRNAs) are large (>200 nucleotides) RNA molecules that make up a significant portion of the non-coding transcriptome and are important regulators of gene expression. We identified a panel of significantly upregulated lncRNAs through analysis of a colon adenocarcinoma RNA-seq data set. These lncRNAs have been associated with tumor progression and metastasis in a number of different cancers. Our aim was to validate expression of these CRC-associated lncRNAs in fresh frozen CRC tissue and normal adjacent epithelium obtained from patients undergoing initial surgical resection.
Methods: CRC and normal adjacent epithelium (>10cm from CRC tissue) from eight patients were acquired from our institution’s tissue biorepository. Tissue specimens were cut, placed onto slides, and stained. Cancer and normal epithelial cells of interest were captured using the ArcturusXT™ Laser Capture Microdissection System, using H&E slides as a reference (Fig. 1A). Total RNA was extracted and expression of lncRNA (FAM83H-AS1, PVT1, UCA1, H19, FER1L4, GAS5, ZFAS1, and GAPDH as internal control) was measured using qRT-PCR.
Results:
In CRC samples, PVT1 & ZFAS1 were significantly upregulated (FC=2.01, p=0.048 & FC= 3.17, p=0.0001, respectively) while FAM83H-AS1 & UCA1 were significantly downregulated (FC=-2.91, p=0.008 & FC=-4.85, 0.002, respectively) compared to normal adjacent epithelium (Fig. 1B). We also observed a significant difference in the expression of PVT1 (p=0.0005) and a trend toward significance in FAM83H in local (Stage I & II) versus metastatic (Stage III & IV) expression (p=0.059)(Fig. 1C). No significant difference was seen in the expression of H19, FER1L4, and GAS5 in CRC samples versus normal adjacent epithelium (Fig. 1B).
Conclusion:
Here we show the differential expression of lncRNAs that are associated with colon adenocarcinoma as compared to normal. PVT1 & ZFAS1 were shown to be significantly upregulated in CRC tissues while FAM83H-AS1 & UCA1 were significantly downregulated. PVT1 was also increased in local compared to metastatic cancers. These results validate the RNA-seq data set for the upregulated lncRNAs but are in contrast to the data set for downregulated lncRNAs. This may be due to a small study population and the use of LCM tissue versus whole tissue, which was used for the RNA-seq data set and has a broader gene expression profile. Additionally, lncRNA primers may not fully capture gene expression. Future studies will verify observed expression profiles in CRC tissues and cell lines via PVT1 & ZFAS1 knockdown and FAM83H-AS1 & UCA1 knock-in experiments.
