Development of a dual-luciferase reporter system for in vivo visualization of MicroRNA biogenesis and posttranscriptional regulation.
MicroRNAs (miRNAs) function in mammalian cells via translational repression or messenger RNA (mRNA) cleavage of target genes by base-pairing with 3' untranslated regions (UTRs) of target mRNAs. Although miRNAs are involved in cell differentiation or organ development, posttranscritptional regulation of miRNA is not well understood. Here, we developed a dual-luciferase reporter system for monitoring in vivo endogenous transcription of primary miRNA (pri-miRNA) and also the mature miRNA activity simultaneously.


METHODS
miR23P639/Fluc plasmid carrying firefly luciferase (Fluc) under the control of miR-23a promoter was used to monitor the transcriptional level of miR-23a, and a cytomegalovirus (CMV)/Gluc/3xPT_mir23 recombinant containing 3 copies of the target sequence of miR-23a in the 3' UTR of Gaussia luciferase (Gluc) before the poly(A) tail was used to monitor the targeting activity of mature miR-23a. This dual-luciferase reporter system transfected to the same population of cells was used to monitor the increased transcriptional level of the pri-miR-23a reflected in the Fluc activity and the decreased Gluc activity affected by mature miR-23a action. Fluc and Gluc activities were also imaged in vivo using the respective substrates in grafted cells in the same nude mice using an in vivo bioluminescence imager.


RESULTS
In HeLa cells and undifferentiated P19 cells, the increased Fluc activity representing the primary miR-23a transcript level reflected the resultant increase in repression of Gluc activity representing mature miR-23a activity. However, 293 cells showed Gluc activity was not repressed as much as Fluc activity was increased, suggesting a block in the posttranscriptional processing of miR-23a transcript in 293 cells. The miR-23a expression in P19 cells before and after neuronal differentiation with retinoic acid treatment showed an increase in Fluc activity and a concomitant decrease in Gluc activity in vitro. HeLa, 293 cells and undifferentiated P19 cells grafted to the nude mice showed exactly the same pattern of luciferase activities in vivo and in vitro.


CONCLUSIONS
We developed a dual-luciferase reporter system to monitor expression and posttranscriptional regulation of a miR-23a in cells in vitro and in vivo. This dual-luciferase reporter system is intended to be used to monitor the expression and regulation of miRNAs noninvasively, especially to understand the differentiation of grafted cells in vivo.
Lee
Ji Young
JY
Interdisciplinary Program in Brain Science, Seoul National University, Seoul, Korea.
Kim
Soonhag
S
Hwang
Do Won
DW
Jeong
Jae Min
JM
Chung
June-Key
JK
Lee
Myung Chul
MC
Lee
Dong Soo
DS
D000818:Animals; D002460:Cell Line; D005786:Gene Expression Regulation / Q000502:physiology*; D017930:Genes, Reporter; D008156:Luciferases / Q000235:genetics; D035683:MicroRNAs / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods*; D009474:Neurons / Q000166:cytology / Q000502:physiology*; D015202:Protein Engineering / Q000379:methods; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D051381:Rats
D035683:MicroRNAs; D008156:Luciferases