Using deep sequencing of formalin-fixed paraffin embedded (FFPE) rectal cancer biopsy tissue to analyze miRNA expression in patients with rectal cancer.

Abstract

487 Background: miRNAs have been shown to be involved in tumor initiation, progression and metastasis in many cancers including colorectal cancer. However, miRNA profiling specifically in rectal cancer is not well characterized. Our objective was to generate a miRNA expression profile in locally advanced rectal cancer using formalin fixed paraffin embedded (FFPE) biopsy tissue collected from patients with locally advanced rectal cancer. Methods: We collected pre-treatment biopsy tissue and matched normal tissue from 40 rectal cancer patients treated with pre-operative chemoradiation (CRT) and total mesorectal excision (TME). We extracted 50-1000ng of total RNA from FFPE biopsies and optimized small RNA sample preparation for deep-sequencing. We then performed deep sequencing on biopsy and matched normal tissue and compared miRNA expression in biopsy and normal tissues using paired T-test and multiple testing (Q-bound <0.05) to determine significant miRNA expression changes in rectal cancer. Results: 182 miRNAs were differentially expressed in tumor versus normal tissues (Q-bound <0.05); 15 of these miRNAs showed a greater than 2-fold change in expression in tumor tissue; mir-18a, mir-135b, mir-503, mir-584, mir-106b, mir-224, mir-92a, mir-181d were up-regulated and mir-375, mir-378, mir-378c, mir-137, mir-378, mir-147b, mir-30a were down-regulated. miRNA mir-31 showed the highest up-regulation in tumor tissue (16-fold increase) while mir-215 expression decreased 8-fold in tumor compared to normal tissue. Tumor and normal tissues were completely separated by hierarchical cluster analysis based on their distinctive miRNA profiles. Conclusions: We optimized small RNA sample preparation for deep sequencing of miRNAs, an approach which may be useful for quantifying miRNA expression in tissues with limited starting material. We also identified a novel miRNA expression profile in rectal cancer that may be useful as a diagnostic biomarker of disease.