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. 2009 Jun;27(6):549-55.
doi: 10.1038/nbt.1543.

Transfection of small RNAs globally perturbs gene regulation by endogenous microRNAs

Aly A Khan  1 , Doron Betel, Martin L Miller, Chris Sander, Christina S Leslie, Debora S Marks
Affiliations

Transfection of small RNAs globally perturbs gene regulation by endogenous microRNAs

Aly A Khan et al. Nat Biotechnol. 2009 Jun.

Erratum in

  • Nat Biotechnol. 2009 Jul;27(7):671

Abstract

Transfection of small RNAs (such as small interfering RNAs (siRNAs) and microRNAs (miRNAs)) into cells typically lowers expression of many genes. Unexpectedly, increased expression of genes also occurs. We investigated whether this upregulation results from a saturation effect--that is, competition among the transfected small RNAs and the endogenous pool of miRNAs for the intracellular machinery that processes small RNAs. To test this hypothesis, we analyzed genome-wide transcript responses from 151 published transfection experiments in seven different human cell types. We show that targets of endogenous miRNAs are expressed at significantly higher levels after transfection, consistent with impaired effectiveness of endogenous miRNA repression. This effect exhibited concentration and temporal dependence. Notably, the profile of endogenous miRNAs can be largely inferred by correlating miRNA sites with gene expression changes after transfections. The competition and saturation effects have practical implications for miRNA target prediction, the design of siRNA and short hairpin RNA (shRNA) genomic screens and siRNA therapeutics.

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Figures

Figure 1
Figure 1. Competition for small RNA machinery
A schematic of the hypothesis that there is competition for RISC machinery between transfected si/miRNA and the cell’s endogenous miRNAs. a. Genes with sites (red) for the transfected small RNA (red) are downregulated after transfection; Genes with sites for endogenous miRNAs (green) may be upregulated after small RNA transfections. b. Biogenesis of miRNAs to show where sh- and siRNAs enter the miRNA processing pathway.
Figure 2
Figure 2. Genes with predicted target sites for endogenous miRNAs are significantly dysregulated after si/miRNA transfections
Visual representation of gene sets tested for significance in expression changes against baseline (B). X is the set of genes with (predicted) sites for exogenous si/miRNAs, D–X is the set of genes with (predicted) sites for endogenous miRNA and no (predicted) sites for exogenous si/miRNAs. p-values shown are calculated by one-sided KS test as described in Methods. a. miR-124 transfection results in up-shift in gene expression for D–X with respect to baseline. b. Pooled data from 15 miRNA transfections into HeLa cells; miR-373, miR-124, miR-148b, miR-106b, miR-124, mut9-10, miR-1, miR-181, chimiR-124-1, chimiR-1-124, miR-16, mir-34a, mir-34b, miR-128a, and miR-9. c. Protein expression changes after let-7b transfection. d. mRNA expression changes after MAPK14-siRNA is transfected into HeLa cells showing upregulation of genes with sites for endogenous miRNAs (green line) [19]; e., f., Genes which contain sites for both endogenous miRNA and transfected small RNAs are less downregulated than if they contain only sites for transfected small RNAs. Changes in gene expression after miR-16 transfections, where p-values shown are calculated by one-sided KS test as described in Methods. e. X–D is the set of genes containing exogenous sites and no endogenous sites. X∩D is the set of genes with sites for both endogenous and exogenous miRNAs. f. X–D≥2 is the set of genes containing exogenous sites but less than 2 endogenous sites. X∩D is the set of genes with sites for both endogenous and exogenous miRNAs.
Figure 3
Figure 3. Quantitative model predicting expression change after transfection
a. Linear regression fitting mean log(expression change) to number of endogenous sites in genes having no exogenous sites, in miR-373 and VHL-siRNA experiments (respectively). One standard deviation from mean depicted with error bars. b. Cartoon graph of genes with different combinations of sites showing net effect on gene expression. c. The relative rank of the ten miRNAs that occurred most often with positive regression coefficient in stepwise regression models for >20 HeLa and 16 HCT116 Dicer−/− transfection experiments (see Methods). These miRNAs can be interpreted as a predicted endogenous profile. The true endogenous HeLa and HCT116 Dicer−/− profile is shown, with miRNAs ranked in order of endogenous expression. Correctly predicted endogenous miRNAs are shown in green and other endogenous miRNAs in black-grey; white indicates a miRNA predicted through regression analysis and not expressed endogenously.
Figure 4
Figure 4. siRNA transfections result in upregulation of genes with sites for endogenous miRNAs, have a dose-response and dynamics similar to targeted effect but in opposite direction
b. A bar graph of the dose of siRNA transfection ranging 0.16nM–100nM versus mean log(expression change) in XNC(grey) and 90th percentile of DNC ≥2 – XNC (green). The graph shows that the putative saturation effect on the endogenous target genes has a dose response similar to the siRNA targeted genes but in the opposite direction. c. A bar graph of mean log(expression change) of XNC(grey) and 90th percentile of DNC ≥2 – XNC (green) versus time. d. A bar graph of log(expression change) versus putative endogenously regulated genes SCML2, TNRC6, YOD1, CX3CL1, AKAP12, and PGM2L1, and a set of MAPK14-siRNA (‘off-targets’) MAPK14 (light-green), MARK2, SLC35F3, HMGB3, FZD7, RPA2, IER5L, at a series of time points over 3 days (genes displayed in this order, from left to right).
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