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RILES, a novel method for temporal analysis of the in vivo regulation of miRNA expression.

Ezzine S, Vassaux G, Pitard B, Barteau B, Malinge JM, Midoux P, Pichon C, Baril P - Nucleic Acids Res. (2013)

Bottom Line: Bioluminescence experiments demonstrated robust qualitative and quantitative data that correlate with the miRNA expression pattern detected by quantitative RT-PCR (qPCR).We further demonstrated that the regulation of miRNA-206 expression during the development of muscular atrophy is individual-dependent, time-regulated and more complex than the information generated by qPCR.As RILES is simple and versatile, we believe that this methodology will contribute to a better understanding of miRNA biology and could serve as a rationale for the development of a novel generation of regulatable gene expression systems with potential therapeutic applications.

View Article: PubMed Central - PubMed

Affiliation: Centre de Biophysique Moléculaire, CNRS UPR4301, Université d'Orléans and Inserm, Orléans, France, UMRE 4320, Faculté de Médecine, Université de Nice-Sophia-Antipolis, Nice, France, Inserm UMR 1087/CNRS UMR 6291, Université de Nantes, Faculté de médecine, L'institut du Thorax, Nantes F-44000 and In-Cell-Art, Nantes F44200, France.

ABSTRACT
Novel methods are required to investigate the complexity of microRNA (miRNA) biology and particularly their dynamic regulation under physiopathological conditions. Herein, a novel plasmid-based RNAi-Inducible Luciferase Expression System (RILES) was engineered to monitor the activity of endogenous RNAi machinery. When RILES is transfected in a target cell, the miRNA of interest suppresses the expression of a transcriptional repressor and consequently switch-ON the expression of the luciferase reporter gene. Hence, miRNA expression in cells is signed by the emission of bioluminescence signals that can be monitored using standard bioluminescence equipment. We validated this approach by monitoring in mice the expression of myomiRs-133, -206 and -1 in skeletal muscles and miRNA-122 in liver. Bioluminescence experiments demonstrated robust qualitative and quantitative data that correlate with the miRNA expression pattern detected by quantitative RT-PCR (qPCR). We further demonstrated that the regulation of miRNA-206 expression during the development of muscular atrophy is individual-dependent, time-regulated and more complex than the information generated by qPCR. As RILES is simple and versatile, we believe that this methodology will contribute to a better understanding of miRNA biology and could serve as a rationale for the development of a novel generation of regulatable gene expression systems with potential therapeutic applications.

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Luciferase expression in HEK 293 cells transfected with several RILES plasmids. Dose–response study of luciferase expression in HEK 293 cells transfected with (A) pRILES/siRNA tGFP T or (B) pRILES/122T in presence of (A) increasing amounts of siRNA tGFP (pU6/shRNA tGFP) and control siRNA (pU6/shRNA Ctl) or (B) increasing concentrations of synthetic miRNA-122. Selective luciferase expression in HEK 293 cells transfected either with (C) pRILES/122T, (D) pRILES/133T or (E) pRILES/221T in the presence of two concentrations of synthetic miRNA-122, −133 and −221. Forty-eight hours after transfection, luciferase expression in cells was determined and expressed as fold induction relative to control cells transfected with the plasmids alone and set to the arbitral value of 1. Data shown are the mean ±SD of one representative experiment performed in triplicate and reproduced at least three times. Statistics by the two-tailed t-test, *P < 0.05; **P < 0.01, n.s (no statistically significant difference) compared to control cells.
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gkt797-F2: Luciferase expression in HEK 293 cells transfected with several RILES plasmids. Dose–response study of luciferase expression in HEK 293 cells transfected with (A) pRILES/siRNA tGFP T or (B) pRILES/122T in presence of (A) increasing amounts of siRNA tGFP (pU6/shRNA tGFP) and control siRNA (pU6/shRNA Ctl) or (B) increasing concentrations of synthetic miRNA-122. Selective luciferase expression in HEK 293 cells transfected either with (C) pRILES/122T, (D) pRILES/133T or (E) pRILES/221T in the presence of two concentrations of synthetic miRNA-122, −133 and −221. Forty-eight hours after transfection, luciferase expression in cells was determined and expressed as fold induction relative to control cells transfected with the plasmids alone and set to the arbitral value of 1. Data shown are the mean ±SD of one representative experiment performed in triplicate and reproduced at least three times. Statistics by the two-tailed t-test, *P < 0.05; **P < 0.01, n.s (no statistically significant difference) compared to control cells.

Mentions: We first performed a series of in vitro proof of principle experiments to evaluate the specificity and the sensitivity of RILES in response to exogenous expressed RNAi molecules such as siRNA molecules encoded by a shRNA plasmid and synthetic miRNAs. Several RILES plasmids were constructed (Supplementary Table 1) and denoted for example pRILES/siRNA tGFPT or pRILES/122T when the RNAi targeting cassette contained complementary sequences to detect the siRNA tGFP or miRNA-122, respectively. These two RILES plasmids, pRILES/siRNA tGFPT and pRILES/122T, were individually transfected in HEK 293 cells in presence of increasing amounts of the shRNA plasmid (Figure 2A) or synthetic precursor miRNA-122 (Figure 2B). Forty-eight hours later, the luciferase activity in cells was determined and expressed as relative fold of luciferase induction by normalizing the values to cells transfected with the pRILES plasmid alone. As shown in Figure 2A and B, increasing the amount of shRNA tGFP plasmid (Figure 2A) or synthetic miRNA-122 (Figure 2B) in pRILES transfected cells also increased the luciferase fold induction values. A maximum 9-fold (±0.3, n = 3, P ≤ 0.01) luciferase induction was found in response to 50 ng shRNA tGFP plasmid (Figure 2A) and a maximum of 26-fold (±1.2, n = 3, P ≤ 0.01) was detected in response to 40 nM of miRNA-122 (Figure 2B). In the latter, the luciferase fold induction was found well correlated (R2 = 0.9321) with the concentration of miRNA ranging from 0 to 20 nM. Overall, our experiments indicated that RILES is able to detect 1 nM of synthetic precursor miRNA molecules in transfected cells; this may represent the detection limit of RILES. It is worth noting that when the second generation of synthetic miRNA molecules (miR Mimics) was used, the detection limit of RILES was as low as 0.3 nM (data not shown). To assess the specificity of RILES, the experiments were conducted with a control mismatch shRNA plasmid (Figure 2A) or with irrelevant miRNAs (Figure 2C–E). No significant luciferase induction was detected in these assays. In contrast luciferase induction was detected only in cells transfected with pRILES/122T, pRILES/133T and pRILES/221T in presence of the corresponding miRNA-122, miRNA-133 and miRNA-221 (Figure 2C–E). We also found that three different synthetic miRNAs investigated at the same concentration were all equally efficient in switching-ON the configuration of RILES and inducing the same level of luciferase gene expression (data not shown). We finally assessed whether RILES could distinguish two closely related miRNA sequences that differ by two nucleotides such as two members of the miRNA-200 family. Results indicated that the pRILES/200cT did not distinguish these two miRNA sequences (Supplementary Figure S1). Next we wanted to determine whether RILES could also monitor the expression pattern of endogenous expressed miRNAs from established cell lines. For this purpose, we exploited the fact that HUH7 and HLE cell lines express opposite levels of miRNA-122 (32) and miRNA-221 (33). These cell lines were transfected individually with the pRILES/122T and pRILES/221T, and luciferase induction was determined 3 days later by normalizing the luciferase values to those found in cells transfected with the control untargeted miRNA RILES plasmid (pRILES). As shown in Supplementary Figure S2, the luciferase induction pattern detected in these cells was found to be remarkably similar to the miRNA expression pattern measured by quantitative RT-PCR. MiRNA-122 and miRNA-221 were oppositely expressed in HUH7 and HLE cells, while miRNA-133 was not significantly detected. Similar specificity of data was also found in C2C12 myoblast cells differentiated in myotubes in vitro to induce expression of the muscle-specific miRNA-133 (34). Again data from quantitative RT-PCR and bioluminescence analysis indicated a similar miRNA expression pattern, i.e. expression of miRNA-133 and almost undetectable expression of miRNA-122 and -221 in differentiated C2C12 cells (Supplementary Figure S2). Remarkably, RILES was also found to be functional in primary hard-to-transfect cells. The luciferase fold induction in primary culture of human dermal fibroblast NHDF was similar to the endogenous expression pattern of miRNA detected by quantitative PCR (data not shown).Figure 2.


RILES, a novel method for temporal analysis of the in vivo regulation of miRNA expression.

Ezzine S, Vassaux G, Pitard B, Barteau B, Malinge JM, Midoux P, Pichon C, Baril P - Nucleic Acids Res. (2013)

Luciferase expression in HEK 293 cells transfected with several RILES plasmids. Dose–response study of luciferase expression in HEK 293 cells transfected with (A) pRILES/siRNA tGFP T or (B) pRILES/122T in presence of (A) increasing amounts of siRNA tGFP (pU6/shRNA tGFP) and control siRNA (pU6/shRNA Ctl) or (B) increasing concentrations of synthetic miRNA-122. Selective luciferase expression in HEK 293 cells transfected either with (C) pRILES/122T, (D) pRILES/133T or (E) pRILES/221T in the presence of two concentrations of synthetic miRNA-122, −133 and −221. Forty-eight hours after transfection, luciferase expression in cells was determined and expressed as fold induction relative to control cells transfected with the plasmids alone and set to the arbitral value of 1. Data shown are the mean ±SD of one representative experiment performed in triplicate and reproduced at least three times. Statistics by the two-tailed t-test, *P < 0.05; **P < 0.01, n.s (no statistically significant difference) compared to control cells.
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gkt797-F2: Luciferase expression in HEK 293 cells transfected with several RILES plasmids. Dose–response study of luciferase expression in HEK 293 cells transfected with (A) pRILES/siRNA tGFP T or (B) pRILES/122T in presence of (A) increasing amounts of siRNA tGFP (pU6/shRNA tGFP) and control siRNA (pU6/shRNA Ctl) or (B) increasing concentrations of synthetic miRNA-122. Selective luciferase expression in HEK 293 cells transfected either with (C) pRILES/122T, (D) pRILES/133T or (E) pRILES/221T in the presence of two concentrations of synthetic miRNA-122, −133 and −221. Forty-eight hours after transfection, luciferase expression in cells was determined and expressed as fold induction relative to control cells transfected with the plasmids alone and set to the arbitral value of 1. Data shown are the mean ±SD of one representative experiment performed in triplicate and reproduced at least three times. Statistics by the two-tailed t-test, *P < 0.05; **P < 0.01, n.s (no statistically significant difference) compared to control cells.
Mentions: We first performed a series of in vitro proof of principle experiments to evaluate the specificity and the sensitivity of RILES in response to exogenous expressed RNAi molecules such as siRNA molecules encoded by a shRNA plasmid and synthetic miRNAs. Several RILES plasmids were constructed (Supplementary Table 1) and denoted for example pRILES/siRNA tGFPT or pRILES/122T when the RNAi targeting cassette contained complementary sequences to detect the siRNA tGFP or miRNA-122, respectively. These two RILES plasmids, pRILES/siRNA tGFPT and pRILES/122T, were individually transfected in HEK 293 cells in presence of increasing amounts of the shRNA plasmid (Figure 2A) or synthetic precursor miRNA-122 (Figure 2B). Forty-eight hours later, the luciferase activity in cells was determined and expressed as relative fold of luciferase induction by normalizing the values to cells transfected with the pRILES plasmid alone. As shown in Figure 2A and B, increasing the amount of shRNA tGFP plasmid (Figure 2A) or synthetic miRNA-122 (Figure 2B) in pRILES transfected cells also increased the luciferase fold induction values. A maximum 9-fold (±0.3, n = 3, P ≤ 0.01) luciferase induction was found in response to 50 ng shRNA tGFP plasmid (Figure 2A) and a maximum of 26-fold (±1.2, n = 3, P ≤ 0.01) was detected in response to 40 nM of miRNA-122 (Figure 2B). In the latter, the luciferase fold induction was found well correlated (R2 = 0.9321) with the concentration of miRNA ranging from 0 to 20 nM. Overall, our experiments indicated that RILES is able to detect 1 nM of synthetic precursor miRNA molecules in transfected cells; this may represent the detection limit of RILES. It is worth noting that when the second generation of synthetic miRNA molecules (miR Mimics) was used, the detection limit of RILES was as low as 0.3 nM (data not shown). To assess the specificity of RILES, the experiments were conducted with a control mismatch shRNA plasmid (Figure 2A) or with irrelevant miRNAs (Figure 2C–E). No significant luciferase induction was detected in these assays. In contrast luciferase induction was detected only in cells transfected with pRILES/122T, pRILES/133T and pRILES/221T in presence of the corresponding miRNA-122, miRNA-133 and miRNA-221 (Figure 2C–E). We also found that three different synthetic miRNAs investigated at the same concentration were all equally efficient in switching-ON the configuration of RILES and inducing the same level of luciferase gene expression (data not shown). We finally assessed whether RILES could distinguish two closely related miRNA sequences that differ by two nucleotides such as two members of the miRNA-200 family. Results indicated that the pRILES/200cT did not distinguish these two miRNA sequences (Supplementary Figure S1). Next we wanted to determine whether RILES could also monitor the expression pattern of endogenous expressed miRNAs from established cell lines. For this purpose, we exploited the fact that HUH7 and HLE cell lines express opposite levels of miRNA-122 (32) and miRNA-221 (33). These cell lines were transfected individually with the pRILES/122T and pRILES/221T, and luciferase induction was determined 3 days later by normalizing the luciferase values to those found in cells transfected with the control untargeted miRNA RILES plasmid (pRILES). As shown in Supplementary Figure S2, the luciferase induction pattern detected in these cells was found to be remarkably similar to the miRNA expression pattern measured by quantitative RT-PCR. MiRNA-122 and miRNA-221 were oppositely expressed in HUH7 and HLE cells, while miRNA-133 was not significantly detected. Similar specificity of data was also found in C2C12 myoblast cells differentiated in myotubes in vitro to induce expression of the muscle-specific miRNA-133 (34). Again data from quantitative RT-PCR and bioluminescence analysis indicated a similar miRNA expression pattern, i.e. expression of miRNA-133 and almost undetectable expression of miRNA-122 and -221 in differentiated C2C12 cells (Supplementary Figure S2). Remarkably, RILES was also found to be functional in primary hard-to-transfect cells. The luciferase fold induction in primary culture of human dermal fibroblast NHDF was similar to the endogenous expression pattern of miRNA detected by quantitative PCR (data not shown).Figure 2.

Bottom Line: Bioluminescence experiments demonstrated robust qualitative and quantitative data that correlate with the miRNA expression pattern detected by quantitative RT-PCR (qPCR).We further demonstrated that the regulation of miRNA-206 expression during the development of muscular atrophy is individual-dependent, time-regulated and more complex than the information generated by qPCR.As RILES is simple and versatile, we believe that this methodology will contribute to a better understanding of miRNA biology and could serve as a rationale for the development of a novel generation of regulatable gene expression systems with potential therapeutic applications.

View Article: PubMed Central - PubMed

Affiliation: Centre de Biophysique Moléculaire, CNRS UPR4301, Université d'Orléans and Inserm, Orléans, France, UMRE 4320, Faculté de Médecine, Université de Nice-Sophia-Antipolis, Nice, France, Inserm UMR 1087/CNRS UMR 6291, Université de Nantes, Faculté de médecine, L'institut du Thorax, Nantes F-44000 and In-Cell-Art, Nantes F44200, France.

ABSTRACT
Novel methods are required to investigate the complexity of microRNA (miRNA) biology and particularly their dynamic regulation under physiopathological conditions. Herein, a novel plasmid-based RNAi-Inducible Luciferase Expression System (RILES) was engineered to monitor the activity of endogenous RNAi machinery. When RILES is transfected in a target cell, the miRNA of interest suppresses the expression of a transcriptional repressor and consequently switch-ON the expression of the luciferase reporter gene. Hence, miRNA expression in cells is signed by the emission of bioluminescence signals that can be monitored using standard bioluminescence equipment. We validated this approach by monitoring in mice the expression of myomiRs-133, -206 and -1 in skeletal muscles and miRNA-122 in liver. Bioluminescence experiments demonstrated robust qualitative and quantitative data that correlate with the miRNA expression pattern detected by quantitative RT-PCR (qPCR). We further demonstrated that the regulation of miRNA-206 expression during the development of muscular atrophy is individual-dependent, time-regulated and more complex than the information generated by qPCR. As RILES is simple and versatile, we believe that this methodology will contribute to a better understanding of miRNA biology and could serve as a rationale for the development of a novel generation of regulatable gene expression systems with potential therapeutic applications.

Show MeSH
Related in: MedlinePlus