MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells

Abstract

MicroRNAs are predicted to regulate thousands of mammalian genes, but relatively few targets have been experimentally validated and few microRNA loss-of-function phenotypes have been assigned. As an alternative to chemically modified antisense oligonucleotides, we developed microRNA inhibitors that can be expressed in cells, as RNAs produced from transgenes. Termed 'microRNA sponges', these competitive inhibitors are transcripts expressed from strong promoters, containing multiple, tandem binding sites to a microRNA of interest. When vectors encoding these sponges are transiently transfected into cultured cells, sponges derepress microRNA targets at least as strongly as chemically modified antisense oligonucleotides. They specifically inhibit microRNAs with a complementary heptameric seed, such that a single sponge can be used to block an entire microRNA seed family. RNA polymerase II promoter (Pol II)-driven sponges contain a fluorescence reporter gene for identification and sorting of sponge-treated cells. We envision the use of stably expressed sponges in animal models of disease and development.

Figure 1

Design of microRNA sponges. (a) We constructed GFP sponges by inserting multiple microRNA binding sites into the 3′ UTR of a 2-h destabilized GFP reporter gene driven by the CMV promoter. (b) The imperfect pairing between a microRNA and a sponge with bulged binding sites is diagrammed for miR-21. We designed sponges with a bulge to protect against endonucleolytic cleavage by Argonaute 2. (c) We constructed U6 sponges by subcloning the microRNA binding site region into a vector containing a U6 snRNA promoter with 5′ and 3′ stem-loop elements.

Figure 2

Efficacy of microRNA sponges. (a–c) RLuc activity relative to firefly luciferase activity was assayed in 293T cells 24 h after transfection with RLuc microRNA target reporters, firefly luciferase transfection control and microRNA sponge plasmids. An RLuc target regulated by 7 miR-20 sites was derepressed by GFP sponges and U6 sponges with bulged or perfect binding sites for miR-20 (a). C, CMV sponge; U, U6 sponge. CX, CXCR4 control; 20b, 7 bulged miR-20 sites; 20pf, two perfect miR-20 sites. Bars represent the expression of the miR-20 target relative to an untargeted control reporter. We measured an artificial CXCR4 target reporter with a single bulged binding site in the presence of a control GFP sponge against miR-21 (miR-21 sponge) or a GFP sponge containing seven CXCR4 binding sites (CXCR4 sponge; b). We transfected cells with 20 nM antisense oligonucleotide (2′ O-methyl 20 or LNA 20) or with the CMV bulged sponge against miR-20 (sponge 20; c). Negative controls; mock (no oligonucleotides or sponges), 2′ O-methyl against miR-30, LNA against miR-122, CXCR4 sponge. We performed each experiment at least three times and have shown a representative example. Error bars, s.d; n = 3.

Figure 3

Specificity of microRNA sponges. (a) We assayed RLuc activity relative to firefly luciferase activity in HeLa cells 24 h after transfection with RLuc microRNA target reporters, firefly luciferase transfection control and microRNA sponge plasmids. Targets of miR-20 and miR-21 are specifically derepressed by the corresponding GFP sponge. Bars are normalized to the relative RLuc units of samples treated with the CXCR4 control sponge. (b) We assayed a perfect target reporter of miR-30c in HeLa cells transfected with oligonucleotide or sponge inhibitors of miR-30e. Controls: 2’ O-methyl anti–miR-181, CXCR4 sponge. MicroRNA sequences below show the heptameric seed sequence in bold, with nucleotide differences between the two family members underlined. We performed each experiment at least three times and have shown a representative example. Error bars, s.d; n = 3.

Figure 4

Validation of microRNA targets. (a) We assayed 293T cells transfected with GFP sponges against miR-18, miR-20 or the CXCR4 control by western blot 48 h after transfection. The increase in endogenous E2F1 upon inhibition of miR-18 or miR-20 is shown relative to the control samples loaded at indicated amounts; E2F1 is the ~60 kDa band indicated; the other bands are nonspecific (top). β-actin loading control (bottom). (b) We assayed 293T cells transfected with an RLuc reporter fused to a fragment of the E2F1 UTR spanning two miR-20 sites, firefly luciferase and GFP sponges. Bars represent RLuc units relative to firefly luciferase units. (c) We assayed RLuc activity relative to firefly luciferase activity in 293T cells transfected with an RLuc reporter fused to a fragment of the CD69 UTR containing a predicted miR-20 binding site, firefly luciferase and GFP sponges. We performed each experiment at least three times and have shown a representative example. Error bars, s.d; n = 3.

Figure 5

Effect of sponges on microRNA levels. (a) We transfected 293T cells with sponge plasmids and collected total RNA for northern blot analysis 48 h later. We probed the blot for miR-16 (top; 24-h exposure), then stripped the blot and reprobed it for GFP mRNA, U6 sponge RNA (both 24-h exposure) and a tRNA loading control (3-h exposure; bottom). (b) Quantitation of miR-16 relative to tRNA for each sponge-treated sample. We performed northern blots for miR-16 and miR-20 >10 times in 293T and HeLa cells, and show results from a representative blot.