Optimization and comparison of knockdown efficacy between polymerase II expressed shRNA and artificial miRNA targeting luciferase and Apolipoprotein B100

Abstract

Background: Controlling and limiting the expression of short hairpin RNA (shRNA) by using constitutive or tissue-specific polymerase II (pol II) expression can be a promising strategy to avoid RNAi toxicity. However, to date detailed studies on requirements for effective pol II shRNA expression and processing are not available. We investigated the optimal structural configuration of shRNA molecules, namely: hairpin location, stem length and termination signal required for effective pol II expression and compared it with an alternative strategy of avoiding toxicity by using artificial microRNA (miRNA) scaffolds.

Results: Highly effective shRNAs targeting luciferase (shLuc) or Apolipoprotein B100 (shApoB1 and shApoB2) were placed under the control of the pol II CMV promoter and expressed at +5 or +6 nucleotides (nt) with reference to the transcription start site (TSS). Different transcription termination signals (TTS), namely minimal polyadenylation (pA), poly T (T5) and U1 were also used. All pol II- expressed shRNA variants induced mild inhibition of Luciferase reporters carrying specific targets and none of them showed comparable efficacy to their polymerase III-expressed H1-shRNA controls, regardless of hairpin position and termination signal used. Extending hairpin stem length from 20 basepairs (bp) to 21, 25 or 29 bp yielded only slight improvement in the overall efficacy. When shLuc, shApoB1 and shApoB2 were placed in an artificial miRNA scaffold, two out of three were as potent as the H1-shRNA controls. Quantification of small interfering RNA (siRNA) molecules showed that the artificial miRNA constructs expressed less molecules than H1-shRNAs and that CMV-shRNA expressed the lowest amount of siRNA molecules suggesting that RNAi processing in this case is least effective. Furthermore, CMV-miApoB1 and CMV-miApoB2 were as effective as the corresponding H1-shApoB1 and H1-shApoB2 in inhibiting endogenous ApoB mRNA.

Conclusion: Our results demonstrate that artificial miRNA have a better efficacy profile than shRNA expressed either from H1 or CMV promoter and will be used in the future for RNAi therapeutic development.

Figure 1

Structure and knockdown efficacy of short hairpins targeting luciferase (shLuc) with different TSS and different TTS. (a) Schematic representation of shLuc constructs expressed from H1 and CMV promoters. Different TTS (pA, T5 and U1) are presented. Hairpin location (+5 or +6) is shown with reference to the TSS (set as +1). The shLuc consists of 20 nt perfectly complementary hairpin structure and a loop of 9 nt. (b) Luciferase knockdown by CMV-shLuc with different TSS and different TTS. Renilla and Firefly luciferase were measured two days post-transfection with 2,5 ng Firefly luciferase reporter, 0,5 ng Renilla luciferase and 100 ng shLuc expressing plasmids. Firefly luciferase expression was normalized to Renilla luciferase expression. H1-shLuc was used as a positive control, H1-shGFP and CMV-shGFP served as negative controls and were set at 100%. Data are represented as mean values ± SD from three independent experiments analyzed with the factor correction method [19]. **p < 0.01 versus negative control One-way ANOVA test with Bonferroni post test.

Figure 2

Structure and knockdown efficacy of CMV-shLuc with different stem length and polyadenylation (pA) or U1 transcription termination signals (U1). (a) Predicted stem-loop structure of CMV-shLuc with different stem lengths (20, 21, 25 and 29 bp). Guide strand is highlighted in grey (b) Luciferase knockdown by CMV-shLuc20, CMV-shLuc21, CMV-shLuc25, CMV-shLuc29 with pA transcription termination signal. (c) Luciferase knockdown by CMV-shLuc20, CMV-shLuc21, CMV-shLuc25, CMV-shLuc29 with U1 transcription termination signal. Renilla and Firefly luciferase were measured two days post-transfection with 2,5 ng Firefly luciferase reporter, 0,5 ng Renilla luciferase and 100 ng short hairpin expressing plasmid. Firefly luciferase expression was normalized to Renilla luciferase expression. H1-shLuc was used as a positive control. H1-shScr and CMV-shScr served as negative controls and were set at 100%. Data are represented as mean values + SD from three independent experiments analyzed with the factor correction method [19]. *p < 0.05, **p < 0.01 versus negative control (One-way ANOVA test with Bonferroni post test).

Figure 3

Structure and knockdown efficacy of shRNA and miRNA hairpin constructs targeting luciferase and Apolipoprotein B100 (ApoB). (abc) Predicted stem-loop structures of shRNA and miRNA targeting luciferase: shLuc or miLuc and ApoB: shApoB or miApoB with guide strand highlighted in grey. shRNA structure is described in Figure 1. miApoB consists of pri-mir-155 precursor sequence, where the mature mir-155 sequence was replaced with the target sequence for luciferase or ApoB. ApoB1 and ApoB2 target different sequences in the ApoB gene. (d) Luciferase knockdown by CMV-shLuc and CMV-miLuc. Renilla and Firefly luciferase were measured two days post-transfection with 100 ng shRNA or miRNA expressing plasmid and 2,5 ng Firefly luciferase and 0,5 ng Renilla luciferase. H1-shLuc was used as a positive control. shScr and miScr served as negative controls and were set at 100%. Firefly luciferase expression was normalized to Renilla luciferase expression. Data are represented as mean values ± SD from three independent experiments analyzed with the factor correction method [19] (e) Knockdown of Luc-ApoB1 reporter, containing in its 3’ UTR ApoB1 target sequence, by CMV-shApoB1 and CMV-miApoB1. Experimental setup was as described in (d) H1-shApoB1 was used as a positive control. (f) Knockdown of Luc-ApoB2 reporter, containing in its 3’ UTR ApoB2 target sequence, by CMV-shApoB2 and CMV-miApoB2. Experimental setup was as described in (d) with the exception that in this Renilla luciferase contained target sequence for ApoB2 and its expression was normalized to Firefly luciferase expression H1-shApoB2 was used as a positive control. **p < 0.01 versus negative control (One-way ANOVA test with Bonferroni post test).

Figure 4

Quantification of siRNA molecules expressed from H1-shRNA, CMV-shRNA and CMV-miRNA targeting luciferase and Apolipoprotein B100. (a) Synthetic siRNA standard lines. siRNA- specific small RNA TaqMan was performed with dilution series of synthetic siLuc, siApoB1 or siApoB2 molecules. Based on molecular weight of the synthetic siLuc, siApoB1 and siApoB2, the amount of molecules for each point of standard line was calculated and plotted against CT value. (b) siLuc amplification plot (left panel) and expression in Hek293T cells (right panel). RNA was isolated two days post-transfection with 1 μg H1-shLuc, CMV-shLuc or CMV-miLuc expressing constructs and siLuc-specific small RNA TaqMan was performed. siRNA copy number was calculated using the synthetic RNA oligo standard line as described in (a) (c) siApoB1 amplification plot (left panel) and expression in Hek293T cells (right panel) after transfection with 1 μg H1-shApoB1, CMV-shApoB1 or CMV-miApoB1 expressing constructs. Experimental set up as described in (b) (d) siApoB2 amplification plot (left panel) and expression in Hek293T cells (right panel) after transfection with 1 μg H1-shApoB2, CMV-shApoB2 or CMV-miApoB2 expressing constructs. Experimental set up as described in (b). Amplification data are presented from representative experiment from two independent experiments conducted with two technical replicates. siRNA expression data are represented as mean values ± SD from 2 independent experiments conducted with two technical replicates.

Figure 5

Comparison of endogenous ApoB mRNA knockdown by shApoB1, shApoB2, miApoB1 and miApoB2in Hepa1-6 cells. Endogenous ApoB mRNA knockdown by H1-shApoB1, CMV-shApoB1 and CMV-miApoB1 constructs (b) Endogenous ApoB mRNA knockdown by H1-shApoB2, CMV-shApoB2 and CMV-miApoB2 constructs qRT-PCR was performed two days post-transfection with 1 μg of shRNA or miRNA expressing constructs and ApoB mRNA levels were calculated relative to actin mRNA. H1-shApoB1 and H1-ApoB1 served as positive control. shScr and miScr served as negative controls and were set at 100%. Data are represented as mean values + SD from three independent experiments analyzed with factor correction method [19]. **p < 0.01 versus negative control (One-way ANOVA test with Bonferroni post test).