Abstract
G A A b st ra ct s patterns between normal colonic tissue (N), tubular adenoma (ADT), tubulovillous adenoma (ADTV) and colorectal cancer (CRC) biopsy tissue samples. Furthermore, our aim was to analyze and confirm the expression level of adenoma and CRC related miRNAs in matched plasma samples. METHODS: Sixty fresh frozen colorectal biopsy (n=20 N, n=11 ADT, n= 9 ADTV, n=20 CRC) and 16 matched plasma samples (n=4 N, n=4 ADT, n=4 ADTV, n=4 CRC) were collected and total RNA including miRNA was isolated (High Pure miRNA Isolation Kit, Roche,Germany). MiRNA expression analysis was performed on GeneChip® miRNA 3.0 Arrays (Affymetrix,USA). Then, the results of microarray analysis were confirmed on RT-qPCR arrays (microRNAReady-to-use PCRHuman Panel I+II V2.R; Exiqon, Denmark). The matched plasma samples were also evaluated by the same miRNA expression and RTqPCR arrays. The obtained results were analyzed by Expression Console (Affymetrix,USA). RESULTS: Out of the 1733 detectable number of miRNAs on the array, in normal biopsies 442, in adenomas 460, and in CRC 441 miRNA could be identified. In plasma these were 306, 334, and 321 miRNA, respectively. Interestingly, 12 miRNAs were upregulated (e.g. miR-31 logFC=3, p<0,001) and 11 miRNA were downregulated only (e.g. miR-10b, logFC=1.7 p<0.001) in neoplastic lesions (AD+CRC) compared to N tissue samples. 11 miRNAs showed altered expression between ADT and ADTV (e.g. miR-183 LogFC=1.5 p<0.007). Expression levels of 9 miRNAs were found to be changed between ADT,TV and CRC groups based on tissue biopsy microarray data (e.g. miR-196a logFC=-1.8 p<0.001). Only three miRNA(-31;-4506;-452*) have been found to be differentially expressed in adenoma compared to normal. Significant positive overexpression was observed in tissue (logFC=5 p= 0.003) and in plasma (logFC=0.3 p=0.02) in case of miR-31 in the adenoma cases. Moreover, increased expressions were detected in CRC compared to healthy controls in tissue and plasma levels of miR-187;-675;-3591-3p (p<0.05). The observed miRNA expression changes could be confirmed by RT-qPCR arrays in both plasma and tissue samples. CONCLUSION: A small number (n=23) of miRNA showed characteristic alterations during neoplastic development in biopsy tissue. Our observations suggest that miRNA are also present in the plasma fraction and their expression is positively correlated with matched tissue expression levels. The identified miRNA expression changes are consistent by different methods through the adenoma-dysplasia-carcinoma sequence.
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