FIG 1.

Engagement of miRNA in target RNA repression affects miRNA level in human cells. (A) Downregulation in cellular miRNA levels in the presence of their substrate mRNAs. Shown is a scheme of reporters used for expression of a target message in excess. Representative blots show let-7a or miR-122 and U6 snRNA levels (endogenous control) in human cell lines expressing RL reporters. Quantifications of miRNA levels from multiple experiments are shown (means ± SEM; n = 3 to 5). (B) Relative levels of let-7a and miR-16 in HeLa cells expressing a control or let-7a reporter from the tetracycline-inducible constructs. Real-time quantifications of endogenous miRNA were done after 24 h of induction and were normalized against U6 snRNA. The value obtained with RL-con was set as the unit (means ± SEM; n = 3). (C) Level of miR-122 and miR-24 in Huh7 cells transfected with increasing concentrations (100 ng to 1,000 ng for 10⁶ cells) of in vitro-transcribed RL reporter mRNA with three miR-122 binding sites. Levels of miRNAs in RL-con (1000 ng) (means ± SEM; n = 3) are used as units. (D) No effect of target mRNA on let-7a miRNA length. Effect of transfection of HeLa cells with increasing concentrations of target mRNA expression plasmid on let-7a miRNA levels evident by Northern blotting done with ³²P-labeled anti-let-7a oligonucleotide. No change in miRNA length was observed. The Northern blot data of U6 snRNA were used as loading control. (E and F) Level of reporter mRNAs in HeLa cells expressing control or let-7a target containing mRNAs (E). In same cells, relative levels of pre-let-7a (left) and pri-let-7a (right) were also measured. All estimation was done using qRT-PCR. RL-con-expressing cells were used as a control (means ± SEM; n = 3) (F). (G) Scheme of target mRNA expression constructs used for reversal of miRNA action of endogenous targets (left) and derepression of let-7a Renilla reporters in HeLa cells expressing GFP let-7a reporter (right). Normalized expression levels of different RL reporters were measured in cells coexpressing GFP mRNAs with or without three let-7a target sites. Fold rescue represents the fold increase in activity of each RL reporter estimated by RL protein expressed in GFP-3×bulge-let-7a expressing cells than GFP-con. (H and I) Expression of target mRNA leads to upregulation of endogenous miRNA targets. Fold increases of endogenous target mRNAs in HeLa (H) and Huh7 (I) cells expressing let-7 a (RL-3×bulgeB-let7a or RL-HMGA2) and miR-122 (RL-3×bulge-miR-122) reporter were estimated by real-time quantification against their expression levels in control reporter-transfected cells. For RL-HMGA2, the control was RL-HMGA2-Mut reporter with mutated let-7a binding sites, while RL-con served as the control for the rest. (J) Reduction in miRNA level by its substrate leads to derepression of endogenous targets at the protein level. Expression of a reciprocally regulated indirect miR-122 target, LDL receptor, and the direct target p53 in Huh7 cells expressing reporters with or without miR-122 sites is shown. β-Actin served as a loading control. ns, nonsignificant. *, P < 0.05; **, P < 0.01; ***, P < 0.0001. P values were determined by paired t test.