Limits...
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.

Show MeSH

Related in: MedlinePlus

Kinetics of luciferase expression in the tibialis anterior muscle of immunodeficient mice. Two micrograms of pRILES/133T, pRILES/122T and control pRILES combined with 6 µg of the pQE30 empty expression plasmid were formulated with the 704 amphiphilic block copolymer and intramuscularly injected in the left and right tibialis anterior to transfect the skeletal muscles of nude mice. ROIs covering the lower legs of animals were drawn and light emission was quantified over time, from day 1 (before the intramuscular injection of RILES-derivates plasmids) to day 38, the end point of the assay. (A) Representative bioluminescence images collected at day 12 from three out of five mice per group. (B) Quantification of bioluminescence signals emitted from mice and plotted as a function of time. Error bars in B, mean ± SEM (n = 6) of one representative experiment repeated two times. Statistics by the two-tailed t-test, *P < 0.05; **P < 0.01 compared with the pRILES control group.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3814383&req=5

gkt797-F5: Kinetics of luciferase expression in the tibialis anterior muscle of immunodeficient mice. Two micrograms of pRILES/133T, pRILES/122T and control pRILES combined with 6 µg of the pQE30 empty expression plasmid were formulated with the 704 amphiphilic block copolymer and intramuscularly injected in the left and right tibialis anterior to transfect the skeletal muscles of nude mice. ROIs covering the lower legs of animals were drawn and light emission was quantified over time, from day 1 (before the intramuscular injection of RILES-derivates plasmids) to day 38, the end point of the assay. (A) Representative bioluminescence images collected at day 12 from three out of five mice per group. (B) Quantification of bioluminescence signals emitted from mice and plotted as a function of time. Error bars in B, mean ± SEM (n = 6) of one representative experiment repeated two times. Statistics by the two-tailed t-test, *P < 0.05; **P < 0.01 compared with the pRILES control group.

Mentions: As miRNA-133 is constitutively expressed in the adult stage of skeletal muscles (34), we conducted a bioluminescence kinetic analysis of miRNA-133 expression in the tibialis anterior muscle of the mice. pRILES/133T, with pRILES/122T and pRILES as controls, was formulated with the 704 copolymer and then intramuscularly injected in the tibialis anterior muscle of mice. The bioluminescence signals emitted from the lower legs of animals were collected at several time points and expressed as the mean of bioluminescence as a function of time (day). As shown in Supplementary Figure S4, a peak of bioluminescence was detected for each group of mice 3 days after the intramuscular administration. Then a stable and constant bioluminescence signal ranging from day 5 to day 15 was observed irrespective of the group of mice. At this plateau, a statistically significant difference (P < 0.01) in luciferase expression was detected in the pRILES/133T versus pRILES/122T and pRILES groups of mice. However, after day 15, bioluminescence signals in the pRILES/133T group rapidly decreased to finally become undetectable at day 20. To determine whether this loss of bioluminescence signal may result from a nonspecific regulation of the inducible promoter, we re-administered the same amount of RILES derivated plasmids and collected the bioluminescence signals subsequently. The bioluminescence signals in mice were lower than those obtained after the first injection and were not statistically different between the various groups of mice (Supplementary Figure S4). We conducted similar experiments in immunodeficient mice to determine whether the loss of luciferase expression was finally related to the development of an immune response directed against either or both the bacterial origin of the CymR repressor or the luciferase protein. In these mice, we found (Figure 5) that the bioluminescence signals detected at the plateau of expression persisted over time and were stable for at least 34 days (end point of our experiment). These data indicate that using RILES, the expression of miRNAs can be monitored for a long period in immunodeficient mice.Figure 5.


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)

Kinetics of luciferase expression in the tibialis anterior muscle of immunodeficient mice. Two micrograms of pRILES/133T, pRILES/122T and control pRILES combined with 6 µg of the pQE30 empty expression plasmid were formulated with the 704 amphiphilic block copolymer and intramuscularly injected in the left and right tibialis anterior to transfect the skeletal muscles of nude mice. ROIs covering the lower legs of animals were drawn and light emission was quantified over time, from day 1 (before the intramuscular injection of RILES-derivates plasmids) to day 38, the end point of the assay. (A) Representative bioluminescence images collected at day 12 from three out of five mice per group. (B) Quantification of bioluminescence signals emitted from mice and plotted as a function of time. Error bars in B, mean ± SEM (n = 6) of one representative experiment repeated two times. Statistics by the two-tailed t-test, *P < 0.05; **P < 0.01 compared with the pRILES control group.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3814383&req=5

gkt797-F5: Kinetics of luciferase expression in the tibialis anterior muscle of immunodeficient mice. Two micrograms of pRILES/133T, pRILES/122T and control pRILES combined with 6 µg of the pQE30 empty expression plasmid were formulated with the 704 amphiphilic block copolymer and intramuscularly injected in the left and right tibialis anterior to transfect the skeletal muscles of nude mice. ROIs covering the lower legs of animals were drawn and light emission was quantified over time, from day 1 (before the intramuscular injection of RILES-derivates plasmids) to day 38, the end point of the assay. (A) Representative bioluminescence images collected at day 12 from three out of five mice per group. (B) Quantification of bioluminescence signals emitted from mice and plotted as a function of time. Error bars in B, mean ± SEM (n = 6) of one representative experiment repeated two times. Statistics by the two-tailed t-test, *P < 0.05; **P < 0.01 compared with the pRILES control group.
Mentions: As miRNA-133 is constitutively expressed in the adult stage of skeletal muscles (34), we conducted a bioluminescence kinetic analysis of miRNA-133 expression in the tibialis anterior muscle of the mice. pRILES/133T, with pRILES/122T and pRILES as controls, was formulated with the 704 copolymer and then intramuscularly injected in the tibialis anterior muscle of mice. The bioluminescence signals emitted from the lower legs of animals were collected at several time points and expressed as the mean of bioluminescence as a function of time (day). As shown in Supplementary Figure S4, a peak of bioluminescence was detected for each group of mice 3 days after the intramuscular administration. Then a stable and constant bioluminescence signal ranging from day 5 to day 15 was observed irrespective of the group of mice. At this plateau, a statistically significant difference (P < 0.01) in luciferase expression was detected in the pRILES/133T versus pRILES/122T and pRILES groups of mice. However, after day 15, bioluminescence signals in the pRILES/133T group rapidly decreased to finally become undetectable at day 20. To determine whether this loss of bioluminescence signal may result from a nonspecific regulation of the inducible promoter, we re-administered the same amount of RILES derivated plasmids and collected the bioluminescence signals subsequently. The bioluminescence signals in mice were lower than those obtained after the first injection and were not statistically different between the various groups of mice (Supplementary Figure S4). We conducted similar experiments in immunodeficient mice to determine whether the loss of luciferase expression was finally related to the development of an immune response directed against either or both the bacterial origin of the CymR repressor or the luciferase protein. In these mice, we found (Figure 5) that the bioluminescence signals detected at the plateau of expression persisted over time and were stable for at least 34 days (end point of our experiment). These data indicate that using RILES, the expression of miRNAs can be monitored for a long period in immunodeficient mice.Figure 5.

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