siRNA-mediated off-target gene silencing triggered by a 7 nt complementation

Abstract

A growing body of evidence suggests that siRNA could generate off-target effects through different mechanisms. However, the full impact of off-target gene regulation on phenotypic induction and accordingly on data interpretation in the context of large-scale siRNA library screen has not been reported. Here we report on off-target gene silencing effects observed in a large-scale knockdown experiment designed to identify novel regulators of the HIF-1 pathway. All of the three 'top hits' from our screen have been demonstrated to result from off-target gene silencing. Two of the three 'siRNA hits' were found to directly trigger down-regulation of hif-1alpha mRNA through a 7 nt motif, AGGCAGT, that is present in both the hif-1alpha mRNA and the siRNAs. Further analysis revealed that the generation of off-target gene silencing via this 7 nt motif depends on the characteristics of the target mRNA, including the sequence context surrounding the complementary region, the position of the complementary region in the mRNA and the copy number of the complementary region. Interestingly, the off-target siRNA against hif-1alpha was also shown to trigger mRNA degradation with high probability of other genes that possess multiple copies of the AGGCAGT motif in the 3'-untranslated region. Lessons learned from this study will be a valuable asset to aid in designing siRNAs with more stringent target selectivity and improving 'hits-follow-up' strategies for future large-scale knockdown experiments.

Figure 1

Off-target silencing of HIF-1α by the original GRK4, HK1 and BTK siRNAs. (A) Left panel: H1299 cells were transfected with the indicated siRNAs using the Lipofectamine 2000 reagent. The expression of GRK4 was determined by QPCR 24 h after transfection. The middle panel: H1299 cells were first transfected with a plasmid encoding a V5-tagged GRK4, then transfected with the indicated siRNAs 24 h later. The cells were collected 24 h after siRNA transfection and analyzed by western blotting using an anti-V5 antibody. Right panel: H1299 cells were first transfected with the dual reporters, pHRE and pRL-TK, then transfected with the indicated siRNAs. Hypoxia treatment was initiated 24 h after siRNA transfection and the cells were collected 12–18 h later for analysis using the Dual-Glo luciferase assay. (B) The effects of the control siRNA, HK1(O) and HK1(N) on the mRNA and protein levels HK1 and the HIF-1 reporter activities were examined as in the left, middle and right panels of (A). A plasmid encoding a V5-tagged HK1 was used for the experiment presented in the middle panel. (C) The effects of the control siRNA, BTK(O) and BTK(N) on the protein level of BTK and the HIF-1 reporter activities were determined as in the middle and right panels of (A). A plasmid encoding a V5-tagged BTK was used for the experiment presented in the left panel. In all experiments, the siRNA were transfected using a final concentration of 20 nM. The original GRK4, HK1 and BTK siRNAs were designated as GRK4(O), HK1(O) and BTK(O). The new GRK4, HK1 and BTK siRNAs were designated as GRK4(N), HK1(N) and BTK(N). Con and HIF-1α represent the control siRNA and the HIF-1α siRNA, respectively.

Figure 2

The original GRK4 and BTK siRNAs down-regulates HIF-1α at both protein and mRNA levels. (A) H1299 cells were transfected with indicated siRNAs for 24 h and subjected to 100 μM DFO treatment (+) or mock treatment (−) for an additional 6 h. The cells were collected and analyzed by western blotting using antibodies against HIF-1α, p53 and β-actin. (B) H1299 cells were transfected with indicated siRNAs, collected 24 h after transfection and analyzed by QPCR for HIF-1α expression. In all experiments, the siRNA were transfected using a final concentration of 20 nM. The original GRK4 and BTK siRNAs were designated as GRK4(O) and BTK(O). The new GRK4 and BTK siRNAs were designated as GRK4(N) and BTK(N). Con and HIF-1α represent the control siRNA and the HIF-1α siRNA, respectively.

Figure 3

The 7 nt motif in GRK4(O)/BTK(O) is responsible for the off-target silencing of HIF-1α. (A) Sequence alignment of the original GRK4 and BTK siRNAs, the two 7 nt boxes (HifBox1 and HifBox2) in the hif-1α mRNA that is complementary to the original GRK4 and BTK siRNAs and the mutated GRK4 siRNA, BTK siRNA, HifBox1 and HifBox2 that carry a C to G change to disrupt the ‘AGGCAGT’ motif. GRK4(O) and BTK(O) represent the original GRK4 and BTK siRNAs in the kinase siRNA library. GRK4(M) and BTK(M) represent siRNAs with a C to G mutation as compared to GRK4(O) and BTK(O). HifBox1m1 and HifBox2m2 represent mutated sequences of the HifBox1 and HifBox2 (each with a C to G mutation compared to HifBox1 and HifBox2). (B) H1299 cells were transfected with indicated siRNAs and collected 24 h later for QPCR analysis of HIF-1α expression. (C) H1299 cells were transfected with indicated siRNAs for 24 h, treated with 100 μM DFO for 6 h and analyzed for HIF-1α expression using western blotting. (D) H1299 cells were transfected with the dual reporters, pHRE and pRL-TK, transfected with indicated siRNAs 24 h after reporter transfection and subjected to hypoxia treatment for an additional 18 h. Cells were then collected and analyzed using the Dual-Glo luciferase assay system. In all experiments, the siRNAs were transfected using a final concentration of 20 nM.

Figure 4

Schematics of luciferase reporters contain different fragments/mutations of the hif-1α mRNA. The luciferase coding region is black colored. The hif-1α mRNA is gray colored. ‘TGA’ is the stop codon of the hif-1α mRNA (2763–2765 nt). The numbers are based on the hif-1α mRNA entry NM_001530. The black boxes represent the two 7 nt boxes, Hif1Box1 and Hif1Box2. Luc-Hif-1α (ORF), Luc-Hif-1α (FL), Luc-Hif-1α (3′UTR) contain the open reading frame of the hif-1α mRNA, the full-length hif-1α mRNA (from the start ATG to the end) and the 3′-UTR of the hif-1α mRNA, respectively. Luc-Hif-1α (3′UTR)m1, Luc-Hif-1α (3′UTR)m2 and Luc-Hif-1α (3′UTR)m3 contain mutations that disrupt the first, second or both 7 nt boxes in the 3′-UTR of the hif-1α mRNA. In Luc-Hif-1α (3′UTR)Box1U200, Luc-Hif-1α (3′UTR)Box1U400, Luc-Hif-1α (3′UTR)Box1D200 and Luc-Hif1α (3′UTR)Box1D400, the first 7 nt box, HifBox1, was moved upstream 200–400 bp, or downstream 200–400 bp from its original position. In the Luc-Hif-1α (ORF)Box1/2 reporter, a 50 bp fragment spanning the two 7 nt boxes in the hif-1α 3′-UTR was inserted into the hif-1α open reading frame. The distance from the stop codon of the luciferase coding sequence to the two 7 nt boxes was kept the same in the Luc-Hif-1α(ORF)Box1/2 reporter as in the Luc-Hif-1α (3′UTR) reporter.

Figure 5

Responses of HIF-1α mutants to gene silencing induced by GRK4(O). (A)–(D) H1299 cells were first transfected with indicated siRNAs and the indicated luciferase reporter variants were transfected together with the pRL-SV40 reporter 24 h after siRNA transfection. The cells were collected and analyzed 18 h later for luciferase activity using the Dual-Glo luciferase assay system. In all experiments, siRNAs were transfected at a final concentration of 20 nM. Con, Luc, HIF-1α and GRK4(O) represent the control siRNA, the siRNA against luciferase, HIF-1α and the original GRK4 siRNA, respectively.

Figure 6

Off-target silencing of additional genes by GRK4(O). H1299 cells were transfected with a control siRNA, GRK4(O), or GRK4(N) at a final concentration of 20 nM. After transfection of siRNA for 24 h, the total RNAs were isolated from the transfected cells and the expression level of each gene was determined by QPCR analyses. The expression level of each gene was first normalized to the expression level of an internal control gene, GADPH and then normalized to its own expression level in cells transfected with the control siRNA. The number in columns label ‘GRK4(O)’ or ‘GRK4(N)’ represents the expression level for each gene in cells transfected with GRK4(O) or GRK4(N). Each number represents data from triplicate transfection (samples from each transfection were run as triplets in the QPCR analysis). An ‘X’ in columns labeled ‘3′UTR’ or ‘CDS’ indicates the presence of the 7 nt motif in the 3′-UTR or the coding region of the gene, respectively. The copy number of the 7 nt motif in each gene is presented in the column labeled ‘Copy of AGGCAGT’.