Decrease in cell viability by siRNA and miRNA dependent induction of DTA.

<p>U251 cells were transfected with the indicated construct (50 ng) with (A) or without (B) siRNAs (10pmol). Cell viability was determined by an XTT assay. The XTT viability value of the “No DTA” construct was set as 100% and the viability value obtained for all other constructs were compared to the No DTA level. (A) DTA dependent decrease in cell viability induced by siRNA in HUH7 cells. The siRNA used were: siCont: control siRNA; S4: siRNA S4; S5: siRNA S5. The numbers above the bars indicated the % viability obtained with each siRNA. P-values are indicated for the specific siRNAs. (B) DTA dependent decrease in cell viability induced by miRNA in U251 cells. The “Const DTA” construct had no UIR, with the DTA transgene directly driven by the CMV promoter. Each bar on the X-axis stands for a construct containing different TS region. The “sTS” control was the 4ORF construct with target sites for siRNAs S4 and S5. The miRNAs for which fully matched target sites were included in the TS are indicated below the bars. The relative level of expression of the miRNAs in the cells (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115327#pone-0115327-t002" target="_blank">Table 2</a>) are marked as L (Low) or H (High). The numbers above the bars of the siRNA indicated the % viability obtained with each siRNA. The experiments were repeated 3 and 4 times, respectively, for A and B. Each experiment was done in triplicates. Statistical analysis was done by T-test and the bars represent standard error. P value for the construct with target sites for miRNAs 21-5p, 125b-5p, 1273g-5p was 1.2*e(−10).</p

MicroRNA expression profiling in various cancer cell-lines.

<p>MicroRNA expression profiling was done on the µParaflo Biochip of LC Sciences. The table present fluorescent values obtained for the listed miRNAs. Microarray data were verified by repetition in selected cell-lines and by quantitative RT-PCR (data not shown).</p><p>MicroRNA expression profiling in various cancer cell-lines.</p

Structure of the selected UIR construct.

<p>Each of the 7 ORFs was ∼600 bp long. ORFs 1–4 were in frame 1, ORFS 5–7 were in frame 2, and the DTA transgene ORF was in the 3^rd frame. T1 and T2 are target sites (TS) for siRNAs. In constructs containing target sites for miRNAs there were usually 3 target sites. Full sequence and information on this construct, with target sites for siRNAs S4 and S5 can be found in the supplementary information).</p

Induction of a renilla luciferase transgene by siRNA.

<p>(A) Three siRNA target sites were placed upstream the renilla luciferase reporter gene, without the UIR, and tested for the known standard siRNA activity. (B) Effects of siRNAs on renilla luciferase reporter gene in a construct containing the 4ORF UIR. In (A) the luciferase activity of the control S4 siRNA was set as 100%. In (B) the luciferase activity of the control S3 siRNA was set as 1 and the luciferase activity values obtained with each of the other siRNAs were compared to that. The experiments were repeated 3 times and each experiment was done in triplicates. Statistical analysis was done by T-test and the bars represent standard error. P Values in A are 2*10(−5); 1*10(−5); 0.002 for S3, S5 and S15, respectively. P Values in B are 0.0008; 1*10(−6) for S3 and S5, respectively.</p

The effect of various UIR configurations on the activity of the DTA transgene.

<p>All construct had the CMV promoter before the UIR. REN l.u. = Renilla luciferase light units as determined by a luminometer. DTA activity is determined by dividing the REN l.u value of the “NO DTA” construct with that of each of the other constructs.</p><p>The effect of various UIR configurations on the activity of the DTA transgene.</p

Transgene activation by siRNAs in constructs containing varied numbers of upstream ORFs.

<p>(A) Activity of the constructs in HEK293T cells. (B) Activity of the constructs in HUH7 cells. HEK293T cells (1.1×10⁵) were transfected with the indicated DTA construct (30 ng), a luciferase reporter plasmid (170 ng) and the indicated siRNA (10 pmol). HUH7 cells (8×10⁴) were transfected with the indicated DTA construct (50 ng), a luciferase reporter plasmid (150 ng) and the indicated siRNA (10 pmol). The renilla luciferase activity of the “No DTA” construct was set as 100% and the luciferase activity obtained for all other constructs were compared to the No DTA level. All constructs had the CMV promoter. The “No DTA” construct had the UIR but had no transgene. The “Const DTA” construct had no UIR, with the DTA transgene directly driven by the CMV promoter. The siRNAs used were: Sc: control siRNA; S4: siRNA S4; S5: siRNA S5. The numbers above the bars of the S4 and S5 siRNA indicated the DTA induction level calculated by dividing the luciferase activity of the Sc siRNA with that of the S4 or S5 siRNA of the same set. The experiments were repeated twice and each experiment was done in triplicates. Statistical analysis was done by T-test and the bars represent standard error. P values in A were 0.005; 2*10(−19); 2*10(−21) for 2ORF, 3ORF and 4ORF respectively. P values in B were 0.008; 6*10(−11); 6*10(−12) for 2ORF, 3ORF and 4ORF respectively. In both cases comparison was to the siRNA controls (Sc).</p

Suppression of the activity of the renilla luciferase transgene by the 4ORF UIR.

<p>REN l.u. = Renilla luciferase light units as determined by a luminometer. The fold reduction was determined by dividing the luciferase light units obtained from the pCMV-REN construct by that obtained from the pCMV-4ORF-REN construct.</p><p>Suppression of the activity of the renilla luciferase transgene by the 4ORF UIR.</p

Reciprocal DTA activation by exogenous siRNAs in HEK293T cells.

<p>HEK293T cells (1.1×10⁵) were transfected with the indicated DTA construct (30 ng), a luciferase reporter plasmid (170 ng) and the indicated siRNA (10 pmol). The renilla luciferase activity of the “No DTA” construct was set as 100% and the luciferase activity obtained for all other constructs were compared to the No DTA level. The “Const DTA” construct had no UIR, with the DTA transgene directly driven by the CMV promoter. The siRNA used were: s4: siRNA S4; s5: siRNA S5; s2: siRNA s2; s13: siRNA S13. SiRNAs S2 and S13 served as controls for the 4ORF-TS4-TS5-DTA construct and siRNA S4 and S5 served as controls for the 4ORF-TS2-TS13-DTA construct. The numbers above the bars of the targeted siRNAs indicated the DTA induction level calculated by dividing the luciferase activity of the average of the control siRNAs with that of the indicted targeted siRNA of the same set. The experiment was repeated 3 times and each experiment was done in triplicates. Statistical analysis was done by T-test and the bars represent standard error. P values are <0.002 for S4 and S5 on the left side and S2 and S13 on the right side.</p

A scheme of the construct design aimed at siRNA or miRNA induction of transgene expression.

<p>(A) The design on the DNA level. (B) The construct on the mRNA level following transcription, no transgene expression due to the inhibitory effect of the UIR. (C) The mRNA product following cleavage by siRNA or miRNA. The cleaved product that includes the transgene contains half of the target site (TS), the UIR is cleaved away, and the transgene can be translated.</p

Induction of DTA by endogenous miRNAs.

<p>(A) Experiments in HUH7 cells. (B) Experiments in HepG2 cells. HUH7 cells (8×10⁴) were transfected with the indicated DTA construct (50 ng), a luciferase reporter plasmid (150 ng). HepG2 cells (8×10⁴) were transfected with the indicated DTA construct (175 ng), a luciferase reporter plasmid (25 ng). The renilla luciferase activity of the “No DTA” construct was set as 100% and the luciferase activity obtained for all other constructs were compared to the No DTA level. The “Const DTA” construct had no UIR, with the DTA transgene directly driven by the CMV promoter. Each bar on the X-axis stands for a construct containing different TS region. The “sTS” control was the 4ORF construct with target sites for siRNAs S4 and S5. The miRNAs for which fully matched target sites were included in the TS are indicated below the bars. The relative level of expression of the miRNAs in the cells (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115327#pone-0115327-t003" target="_blank">Table 3</a>) are marked as L (Low), M (Medium) or H (High). The numbers above the bars indicate the fold reduction of luciferase activity (and thus induction of DTA activity) between the relevant construct and the sTS control construct. The experiments were repeated 3 and 5 times, respectively, for A and B. Each experiment was done in triplicates. Statistical analysis was done by T-test and the bars represent standard error. P values in A were P<0.003 for all data points. P value in B were <0.01 for the two constructs containing the miRNAs 21-5p, 125b-5p, 1273g-5p target sites. For the construct containing target sites for miRNAs 9-5p, 15b-5p, 16-5p the p Value was 0.24.</p