Synthetic pre-microRNAs reveal dual-strand activity of miR-34a on TNF-α

Abstract

Functional microRNAs (miRNAs) are produced from both arms of their precursors (pre-miRNAs). Their abundances vary in context-dependent fashion spatiotemporarily and there is mounting evidence of regulatory interplay between them. Here, we introduce chemically synthesized pre-miRNAs (syn-pre-miRNAs) as a general class of accessible, easily transfectable mimics of pre-miRNAs. These are RNA hairpins, identical in sequence to natural pre-miRNAs. They differ from commercially available miRNA mimics through their complete hairpin structure, including any regulatory elements in their terminal-loop regions and their potential to introduce both strands into RISC. They are distinguished from transcribed pre-miRNAs by their terminal 5' hydroxyl groups and their precisely defined terminal nucleotides. We demonstrate with several examples how they fully recapitulate the properties of pre-miRNAs, including their processing by Dicer into functionally active 5p; and 3p-derived mature miRNAs. We use syn-pre-miRNAs to show that miR-34a uses its 5p and 3p miRNAs in two pathways: apoptosis during TGF-β signaling, where SIRT1 and SP4 are suppressed by miR-34a-5p and miR-34a-3p, respectively; and the lipopolysaccharide (LPS)-activation of primary human monocyte-derived macrophages, where TNF (TNFα) is suppressed by miR-34a-5p indirectly and miR-34a-3p directly. Our results add to growing evidence that the use of both arms of a miRNA may be a widely used mechanism. We further suggest that syn-pre-miRNAs are ideal and affordable tools to investigate these mechanisms.

Keywords: 3p strand; TNF-α; macrophages; miR-34a; miRNA biogenesis; pre-miRNA.

FIGURE 1.

MiR-34a-5p and miR-34a-3p show antiproliferative activity. (A) siTGF was transfected into HeLa cells in increasing doses (0, 2, 9, and 36 nM). RNA was isolated and qPCR was performed for miR-34a-5p and miR-34a-3p. The y-axis shows the relative RNA abundance (normalized against miR-16 and an untransfected control). Error bars, SD of four replicates. (B) Endogenous miR-34a-5p and miR-34a-3p, induced during TGFB RNAi, suppress luciferase reporter genes bearing complementary target sites. Transfections of Con (negative control siRNA), siTGF (siRNA against TGFB), and siRen (siRNA against Renilla luciferase) into HeLa cells were performed at increasing concentrations (0, 2, 9, and 36 nM). Luciferase reporter plasmids bearing no (psiCHECK2) or a single reverse complementary target site (TS) for the miRNA (origin of the TS shown in the inset) were transfected. Renilla luminescence values were normalized against firefly luminescence and then to the value from the corresponding 0-nM transfection for each treatment. Error bars, SD of three transfections. (C) MiR-34a-5p and miR-34a-3p, induced during TGFB RNAi, contribute to apoptosis. HeLa cells were transfected with Con (negative control), syn-pre-34a, miRNA mimics 34a-5p and 34a-3p, and siRen at increasing concentrations (0, 2, 9, and 36 nM). Caspase 3/7 activity was measured from lysates of HeLa cells 72 h post-transfection. Error bars, SD of three transfections (*) P < 0.05; (**) P < 0.01; (***) P < 0.001.

FIGURE 2.

Properties of chemically synthesized pre-miRNA mimics. (A) HPLC-MS chromatogram of syn-pre-34a. The x-axis represents column retention time; the y-axis shows UV-peak intensity. (B) Syn-pre-34a was incubated with recombinant Dicer for 2 h at 37°C in buffer. Analysis by HPLC-MS indicates processing of the precursor (slowest migrating product) into distinct product groups: the pre-miR-34a terminal loop, the miR-34a-5p, and miR-34a-3p strands. (C) Table of predicted products generated by Dicer cleavage of syn-pre-34a, including observed molecular masses and predicted molecular masses. (D) Abundance of miR-34a-5p and miR-34a-3p strands after transfection of syn-pre-miR34a into HeLa cells at increasing doses (2, 9, and 36 nM), followed by RNA isolation and qPCR using primers for miR-34a-5p and miR-34a-3p strands. The y-axis shows the relative RNA abundance (normalized against internal miR-16 and an untransfected control). (E) Syn-pre-34a (0, 0.5, 2, 9, and 36 nM) and ds-34a (9 nM) were transfected into HeLa cells. Total RNA was isolated and probed by Northern blotting for miR-34a-5p and miR-34a-3p, as well as U6 snRNA, with radioactively labeled probes.

FIGURE 3.

Syn-pre-miRNAs are processed in cells to functional 5p and 3p miRNAs. (A) Syn-pre-miRNAs and their corresponding miRNA mimics were compared for inhibitory activity in dual-luciferase reporter assays. Transfections of Con, syn-pre-miRNAs, corresponding miRNA mimics, and siRen into HeLa cells were performed at increasing concentrations (0, 2, 9, and 36 nM). Luciferase reporter plasmids bearing a single validated target site (TS) for the miRNA (origin of the TS shown in the inset) were transfected. Renilla luminescence values were normalized against firefly luminescence, and then to the value from the corresponding 0 nM transfection. Error bars, SD of three transfections (*) P < 0.05; (**) P < 0.01; (***) P < 0.001. (B) Syn-pre-miRNAs from the miR-106 family show varying levels of inhibition of luciferase reporters containing target sites from CDKN1A and TGFBR2. Transfections and work-up were performed as in A. Error bars, SD of three transfections (*) P < 0.05; (**) P < 0.01; (***) P < 0.001. (C) Syn-pre-miRNAs (miR-106a, miR-107, miR-34a) are processed to functional 5p and 3p miRNAs in HeLa cells and suppress luciferase reporters containing a single complementary target site. Syn-pre-miRNAs and controls were transfected with increasing concentrations (0, 2, 9, and 36 nM) into HeLa cells. Work-up and data analysis were performed as in A. Error bars, SD of three transfections (*) P < 0.05; (**) P < 0.01; (***) P < 0.001. (D) Sequence homology between the 5p and 3p miR-17 family members.

FIGURE 4.

Identification of new targets for miR-34a-3p. (A) SiTGF and miR-34a-5p repress SIRT1 3′ UTR in a dual-luciferase reporter assay. Transfections of dsRNAs into HeLa cells were performed at increasing concentrations (0, 2, 9, and 36 nM). Luciferase reporter plasmid bearing a single validated target site from SIRT1 (Yamakuchi et al. 2008) for miR-34a-5p was transfected. Renilla luminescence values were normalized against firefly luminescence and then to the value from the corresponding 0-nM transfection. Error bars, SD of three transfections (*) P < 0.05; (**) P < 0.01; (***) P < 0.001. (B) Syn-pre-34a and 34a-5p mimic repress SIRT1 protein in HeLa cells. Cells were transfected with increasing concentrations (0, 2, 9, and 36 nM) of dsRNAs. Total protein was isolated 48 h post-transfection. SIRT1 protein expression was analyzed by Western blot using a specific antibody and was quantified by densitometry using ImageJ (rsbweb.nih.gov/ij/). (C) Syn-pre-34a and 34a-3p mimic repress target sites from SP4, JUN, and TNF 3′ UTRs predicted by the mirSVR algorithm (Betel et al. 2010). Transfections of dsRNAs into HeLa cells were performed at increasing concentrations (0, 2, 9, and 36 nM). Luciferase reporter plasmids (identity shown in inset) bearing either a single predicted target site (SP4 and JUN) or two predicted target sites (TNFα) and their corresponding mutated controls were transfected. Renilla luminescence values were normalized against firefly luminescence and then to the value from the corresponding 0-nM transfection. Error bars, SD of three transfections (*) P < 0.05; (**) P < 0.01; (***) P < 0.001. (D) Syn-pre-34a and 34a-3p repress SP4 protein in HeLa cells. Cells were transfected with increasing concentrations (0, 2, 9, and 36 nM) of dsRNAs. Total protein was isolated 48 h post-transfection. SP4 protein expression was analyzed by Western blot using a specific antibody and was quantified by densitometry using ImageJ.

FIGURE 5.

MiR-34a-5p and MiR-34a-3p strands are active in TNFα signaling. (A) Primary monocyte-derived macrophages from five human donors were stimulated with LPS (100 ng/mL). RNA was isolated and qPCR for miR-34a-5p and miR-34a-3p strands and internal controls (miR-16 and miR-191) was performed. The y-axis shows the relative RNA abundance (normalized against internal miR-16 and an unstimulated control). Error bars, SEM of five donors. (*) P < 0.05; (**) P < 0.01; (***) P < 0.001. (B) Syn-pre-34a was transfected at increasing concentrations (0, 2, and 9 nM) into macrophages. RNA was isolated and qPCR was performed for miR-34a-5p and miR-34a-3p. The y-axis shows the relative RNA abundance (normalized against internal miR-16 and an untransfected control). Error bars, SD of four replicates. (C) Syn-pre-34a, 34a-5p, and 34a-3p (2 and 9 nM), Con (9 nM) and transfection reagent alone (mock) were transfected into macrophages derived from three human donors. Total RNA was isolated and SYBR-Green qPCR was used to assay TNFα mRNA. The y-axis shows C_t-values representing the absolute abundance of the mRNA normalized to GAPDH mRNA, and then to mock control. Error bars, the SD of four replicates. (D) Syn-pre-34a, 34a-5p, and 34a-3p (2, 9, and 36 nM), Con (9 and 36 nM), and transfection reagent alone (mock) were transfected into macrophages derived from three human donors. Cells were stimulated with LPS (100 ng/mL) and secreted TNFα protein was measured by ELISA after 12 h. The y-axis shows amounts of secreted TNFα protein (nanogram/well). The transfection reagent without LPS treatment (mock – LPS) shows background levels of TNFα protein in unstimulated cells. Error bars, the SD of two transfections. (E) Transfection of syn-pre-34a, 34a-5p, and 34a-3p (2, 9, and 36 nM), Con (9 and 36 nM), and transfection reagent alone (mock) were performed in macrophages derived from three donors. Secreted TNFα protein was assayed (0, 3, 8, and 24 h) after stimulation with LPS (100 ng/mL) using ELISA. The y-axis shows relative amounts of secreted TNFα protein. The transfection reagent without LPS stimulation (mock – LPS) shows background levels of TNFα protein prior to stimulation. Error bars, the SD of three donors with duplicate transfections.