Interaction of MMR gene 3'UTRs with microRNAs.

(a) Bar diagram illustrating positive 3'UTR activity of hMLH1, hMSH6 and hMSH2 genes in HEPG2 cells following reporter gene assay. Fold changes were appended in the diagram. (*) indicates statistical significance of functionality of 3'UTR regions measured in terms of P value by t-test. (b) Entire 3'UTR region of hMLH1, hMSH2 and hMSH6 were mapped for putative microRNA binding sites. The highlighted and boldfaced segments within the 3'UTR sequences represent the seed positions for the microRNAs. (c) Multiple alignments indicated that miR-155, miR-21 and miR-141 seed sequences are evolutionarily conserved across mammalian and non-mammalian species. (d) Normalized relative light units (RLU) in HEPG2 and PC3 cells were measured for hMLH1 and hMSH6 3'-UTR constructs in pSiCHECK2 with (+) and without (-) the effect of microRNAs. Co-transfection of the pRNAU6.1 empty vector (+) with the 3'UTR-pSiCHECK2 constructs of hMLH1 and hMSH6 were set as 100% and percentage reduction in luciferase activity mediated by the three microRNAs were measured in relation to this was shown in the bar diagram. Statistical significance in terms of P values was measured with t-test. (*) indicates the statistical significance P<0.05. (e) Bar diagram showing upregulation of hsa-miR-21, hsa-miR-155 and hsa-miR-141transcripts in prostate cancer tissues compared to that of BPH patients. * indicates P<0.05. (f) Heat map showing the combination of two dendrograms displayed above and to the right. The rows represent genes on the right of the figure. Individual patient sample is shown as columns. Color represents expression level of the gene. Red represents low expression, while green represents high expression. The expression levels are continuously mapped on the color scale provided at the bottom of the figure. The dendrograms show stratification of samples into two groups: BPH (B) and prostate cancer (P), based on gene expression.