Assessing the Potential of the Htert Cell Line as a Model of Normal Bladder Urothelium via Analysis of miRNA Expression
Yasmin Akbari, B.A.1, Derek Beaton, M.A.2, Benjamin Keenan, M.A.1, Van Le, B.A.1, Tanmay Parekh, B.A.1, Joon Hee Lee, B.A.1, Travis Antes, PhD3, Louis Liou, M.D./PhD1.
1Boston University, Boston, MA, USA, 2University of Massachusetts, Dartmouth, MA, USA, 3System Biosciences, Mountain View, CA, USA.
BACKGROUND:MicroRNAs (miRNAs) are a recently discovered class of short noncoding RNA molecules. Current research suggests that miRNAs are involved in regulation of human gene expression, notably demonstrating differential expression levels across various stages of cancer progression. A panel of miRNAs that are differentially expressed in malignant tissue can serve as markers for cancer, and can then be altered in cell culture in order to assess their mechanistic validity. We have analyzed the miRNA expression patterns in cell lines of high grade and low grade human urothelial carcinoma (UC), as well as the newly developed Htert cell lines, representative of normal bladder urothelium. By comparing the expression of miRNAs in cancer cell cultures to both the Htert lines as well as normal urothelium cells collected from male urine cytology, we seek to determine the potential of the Htert cell line to serve as a model for normal bladder urothelium.
METHODS: Cells were collected from cell lines and urine cytology representing a total of two sets of 10 patients: 5 high grade UC, 2 low grade UC, and 3 normal urothelium (one set using the Htert lines, the other the normal urothelium). Cell lines expressing high grade (5637 and T24) and low grade (RT4) UC were provided by American Type Culture Collection (Manassas, VA). Htert001 and Htert002 lines were provided by Cleveland Clinic (Cleveland, OH). Normal urothelium cells were collected from urine samples from Boston Medical Center (Boston, MA). IRB approval was obtained from our institution. MiRNAs were extracted using the mirVana miRNA Isolation Kit (Ambion, California) and then quantified and reverse transcribed to cDNA using Quantimir (Systems Bioscience, California). Real-Time PCR was performed in triplicate in 384-well plates using primers from the Oncomir Kit (Systems Bioscience). MiRNA expression data was normalized using U6 as an endogenous control. The two patient sets -- one including the Htert lines, and the other including normal urothelial cells -- were analyzed separately. MiRNAs of interest were selected by performing a two-tailed t-test comparing cancer to normal cells (p<0.05). Hierarchical clustering was used to select those miRNAs that appear to distinguish cancer from normal cells in their relative expression levels..
RESULTS: Five miRNAs in the Htert set and 10 miRNAs in the normal urothelium set demonstrated statistically significant differential expression. 3 miRNAs were common to both sets: hsa-let-7a, hsa-let-7e, and miR-107. Analysis of expression level fold changes of each miRNA were also shown to be comparable relative to other miRNAs.
CONCLUSIONS:Similarities in miRNA expression patterns between the Htert line and normal urothelium suggest that the Htert cell line may be a suitable model for normal urothelium. Further experiments will use the Htert line in transfection studies to assess the biological significance of potential miRNAs found to be differentially expressed in malignant bladder tissue when compared to normal urothelium. These may serve as the basis of diagnostic and/or prognostic markers for UC.
|miRNA||p-value||Fold Change, High Grade||Fold Change, Low Grade|