J. Burton1, J. Carter1, N. Galbraith1, M. R. Eichenberger1, S. Galandiuk1 1University Of Louisville,Department Of Surgery,Louisville, KY, USA
Introduction:
Cell lines are a common experimental model used to study biological mechanisms involved in various diseases. In-vitro cell line studies allow for the investigation of signaling pathways, functional processes, identification of molecular markers of disease, and testing of cancer therapeutics. microRNAs (miRNAs) affect gene expression by negative regulation of target mRNA and are dysregulated in a variety of cancers including colorectal cancer (CRC). They exhibit a variety of regulatory functions related to cell growth, development, and differentiation. miRNAs are inherently stable accounting for their emerging use as biomarkers for human disease and as targets for disease intervention. We chose to characterize the differential expression of miRNAs in CRC as compared to a normal colon epithelial cell line in order to identify potential therapeutic targets for intervention.
Methods:
Sporadic CRC cell lines (SW116, SW480, HT29, T84) representing locally confined to metastatic disease (modified Dukes’ A-D staging) and a normal colon epithelial cell line (CCD841) were acquired (ATCC®, Manassas, VA). Total RNA was extracted from cells and RNA quantity and purity determined. For each cell line, RT and qRT-PCR was performed. The expression levels of 380 miRNAs were examined using microfluidic array technology (Life Technologies, Carlsbad, CA). miRNA expression of each CRC cell line was compared to the miRNA expression of the normal colon epithelial cell line. Statistical analysis was performed using a two sample t-test and results were regarded significant when p<0.05.
Results:
Each cell line was screened 4 times. Comparison of miRNA expression in non-metastatic CRC cell lines (Dukes’ A, SW116, and Dukes’ B, SW480) compared to normal colon epithelium (CCD841) identified 190 significantly dysregulated miRNAs, 36 up-regulated and 139 down-regulated miRNA (p<0.05). Five of the 8 most significantly upregulated miRNAs were members of the miR-200 family (p<0.0001, fold-change 2688-18830). Comparison of metastatic CRC cell lines (Dukes’ C, HT29, and Dukes’ D, T84) as compared to normal colon epithelium identified 231 significantly dysregulated miRNAs, 39 up-regulated and 192 down-regulated miRNA (p<0.05). Five of the 6 most up-regulated miRNAs were all members of the miR-200 family (p<0.0001, fold-change 4171-35668). Single assay confirmation of individual miR-200 family members was performed in each cell line.
Conclusions:
We have characterized the miRNA expression in CRC cell lines as compared to a normal colon epithelial cell line. Identification of dysregulated miRNAs permits identification and investigation of clinically relevant miRNAs as potential targets for disease intervention in CRC. Members of the miR-200 family were highly expressed in all tested CRC cell lines. This provides an opportunity to study the miR-200 family already known to play a role in tumor progression and cancer metastasis.