Figure 2

Accumulation of Cleaved Target kRAS mRNA 5′-fragments by miR-622 Overexpression in H1299 Cells.

(a) miR-622–targeted kRAS mRNA cleavage was detected by SLA–RT-PCR. Total RNAs were isolated from H1299 cells transfected with a miR-622 expression vector or a let-7d expression vector as a nonspecific control. RT reactions were performed using SLA-RT and 2U-SLA-RT primers (Supplementary Table 1d) for detection of cleaved kRAS target mRNA fragments (as indicated by red bars between the matched bases in seed region with and without 3′-uridylation, respectively, SLA–RT-PCR products were analysed by 2% agarose gel electrophoresis and shown in upper panels. M: 0.03 μg of 1 kb DNA ladder. Results of the quantitative real-time RT-PCR (SLA-qRT-PCR) analysis of miR-622-mediated target kRAS mRNA cleavage and uridylation activities at corresponding sites to the above SLA-RT-PCR reactions were represented as relative fragment abundance (RFA) compared to the highest amplicon abundance in individual qRT-PCR series and shown in lower graph panels. The mature cel-miR-67 expressing plasmid was used as a negative control. RFA values were represented as the mean of three independent experiments and error bars as standard errors to the mean. Overexpression of miR-622 resulted in an accumulation of cleaved kRAS mRNA fragments at bases G6, U7 and C8 and of the 3′-oligouridylated fragments at bases G6 and C8 corresponding to the miR-622 seed region but with ignorable cleavage and uridylation activities at bases C12-C20 in central bulge and supplementary base pair regions, as indicated by red boxes. (b) Relative normalised expression (RNE) levels of let-7 miRNAs, miR-622 and miR-30a were determined in H1299 cells transfected with either let-7d or miR-622 expression vector by qRT-PCR.