Fig. 2.

MicroInspector alignment of miRs with KRT_366–1067 and KRT_967–1466 transcripts, and effects of miRs on cellular levels of KRT RNA and protein. Library selection was performed on the KRT segments as described, and the MicroInspector tool was then used to align miRs from the database with the respective segments of KRT. Results for KRT_366–1067 are shown in panel A, and results for KRT_967–1466 are shown in panel B. Library-selected regions are shown at the top of the panels; they are shown as 17 nt regions, based on the library size, although they may actually extend in either direction (for KRT_366–1067, R1–R8, shown in blue, were identified on sequencing gels run for 1.5 h, and R9–R12, shown in red, were identified on gels run for 7.5 h). Eighty percent of the miRs which aligned with KRT_366–1067 overlapped (≥5 nt) empirically identified accessible regions. The percentage of miRs which overlapped with accessible regions in KRT_967–1466 was 76%, including the shift of regions 1 and 7 (Δ1 and Δ7 were shifted by six nucleotides) to compensate for RNase-H hydrolysis initiation. In particular, an astounding 17 aligned miRs overlapped with R9 in KRT_366–1067, (panel A) and 7 aligned miRs overlapped with R7 which lies within the 3′-untranslated region of KRT_967–1466 (panel B; the position of the stop codon is shown in red), with an additional 6 miRs aligned immediately 3′ to R7. MCF7 cells were transfected with the specified miRs, and cells were harvested 48 h later. Panel C: RNA was examined for KRT transcripts using QPCR, with normalization to TBP (the amplicon was KRT_548–648). Additional controls included miRs which did not align with KRT. Panel D: KRT protein expression was examined by immunoblot analysis using an antibody against KRT. β-actin was used as a loading control. This shows results of a representative experiment. Panel E shows densitometric analysis of KRT protein expression levels from three independent experiments, as means ± standard errors. Asterisk (*) indicates significant differences at P<0.01 or greater from controls. For panels C-E: Lanes 1 and 2 show results with miRs which aligned with R1 (miR-125b-1 and miR-615-5p, respectively). Lane 3 shows results with an miR which aligned between R2 and R3 (miR-138). Lane 4 shows results with an miR which aligned with R6 (miR-let-7b). Lanes 5–7 show results with three miRs which aligned with R7 in the3′-untranslated region (miR-205, miR-150, and miR-211, respectively). Lane 8 shows results with untreated MCF7 cells. Transfections without miR, or with a negative control miR which did not align with KRT were also without effect (data not shown; Lanes 5–7 effectively serve as negative controls for the protocol). Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]