HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH RSS TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text Full Text (PDF) Supplemental Data Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in JNM Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Lee, J. Y. Articles by Lee, D. S. Search for Related Content PubMed PubMed Citation Articles by Lee, J. Y. Articles by Lee, D. S.

Development of a Dual-Luciferase Reporter System for In Vivo Visualization of MicroRNA Biogenesis and Posttranscriptional Regulation

¹ Interdisciplinary Program in Brain Science, Seoul National University, Seoul, Korea; ² Department of Nuclear Medicine, Seoul National University College of Medicine, Seoul, Korea; and ³ Medical Research Center, Seoul National University College of Medicine, Seoul, Korea

View larger version (8K): [in this window] [in a new window]   FIGURE 1.  Reporter constructs to monitor primary transcript and mature miRNA activities. (A) miR23P639/Fluc. Segment ranging from 603 to 36 bp upstream to the transcription initiation site of miR23a27a24-2 gene clusters was inserted into pGL3_basic (Promega) containing a promoterless Fluc gene. (B) CMV/Gluc/3xPT_mir23. CMV/Gluc carries 3 copies of target sequence perfectly complementary to miR-23a (3xPT_mir23) in its 3' UTR.

View larger version (15K): [in this window] [in a new window]   FIGURE 2.  Validation of CMV/Gluc/3xPT_mir23 construct to represent mature miR-23a activity. (A) Mature miR-23a sequence and its relevant target sequence, which were inserted into the 3' UTR of Gluc reporter gene. Two targets of 3xPT_mir23 sequences were constructed; one was a perfect complement and the other was an inverted sequence and was used as negative control. (B) Constructs of CMV/pri-miR-23a/Fluc and CMV/Fluc used as exogenous source and its negative control of pri-miR-23a. (C) CMV/Gluc/3xPT_mir23 and CMV/Gluc/3xPT_mir23_inv were transfected into HeLa cells. Endogenous mature miR-23a in HeLa cells repressed Gluc activity of transfected CMV/Gluc/3xPT_mir23 construct but did not repress that of transfected CMV/Gluc/3xPT_mir23_inv construct. (D) CMV/pri-miR-23a/Fluc and CMV/Gluc/3xPT_mir23 were transfected into 293 cells with their respective controls of CMV/Fluc and CMV/Gluc. Exogenously expressed pri-miR-23a should have been processed to mature miR-23a and, thus, repressed Gluc activity partially in 293 cells cotransfected with CMV/pri-miR23a/Fluc and CMV/Gluc/3xPT_mir23. Cotransfected CMV/Fluc, which does not express pri-miR-23a, did not repress Gluc activity of 293 cells transfected with CMV/Gluc/3xPT_mir23. *P < 0.001.

View larger version (9K): [in this window] [in a new window]   FIGURE 3.  Enhancement of Fluc representing endogenous expression of miR-23a and repression of Gluc representing mature miR-23a activity in HeLa and 293 cells. (A) Compared with pGL_basic activity, miR23P639/Fluc transfection revealed 10-fold increase in firefly luciferase activities in HeLa and 293 cells. Increased Fluc activities represented increased expression of endogenous pri-miR-23a in these cells. (B) Compared with CMV/Gluc transfection, CMV/Gluc/3xPT_mir23 transfection revealed decrease of Gaussia luciferase activities in HeLa cells but not in 293 cells. Repressed Gluc activities represented the presence of mature miR-23a activity and, thus, concordant expression of miR-23a in HeLa cells but discordant expression of pri-miR-23a as well as the activity of mature miR-23a in 293 cells. Data are expressed as mean ± SD of triplicates.

View larger version (24K): [in this window] [in a new window]   FIGURE 4.  (A) Levels of endogenous pri-miR-23a were determined by RT-PCR in HeLa and 293 cells. β-Actin was used as control. (B) Relative quantity of mature miR-23a on real-time qRT-PCR in HeLa and 293 cells. Values are mean ± SD of triplicates. U6 snRNA was used as an endogenous control. Ct = threshold cycle (Ct = CtmiRNA – CtU6RNA, Ct293 = Ct293 – Ct293, CtHeLa = CtHeLa – Ct293).

View larger version (39K): [in this window] [in a new window]   FIGURE 5.  Neuronal differentiation of P19 by RA treatment. (A) Sequential phase-contrast images of P19 neurogenesis. Cells developed a neuronal morphology first by rounding up and then by producing protrusions. Finally, cells were organized into neurosphere-like colonies with growing neurites. (B) RNAs were prepared from undifferentiated or differentiated P19 cells on days 3 and 9 of RA treatment and were analyzed by RT-PCR. Oct4 expression was reduced after RA treatment. RA treatment caused P19 cells to differentiate into MAP2-positive neuronal cells.

View larger version (22K): [in this window] [in a new window]   FIGURE 6.  Pri-miR-23a expression and mature miR-23a activities in P19 cells during neurogenesis and confirmation of expression pattern by RT-PCR for primary miR23a and qRT-PCR for mature miR23a. (A) Compared with pGL_basic activites, miR23P639/Fluc transfection increased firefly luciferase activities in undifferentiated P19 cells, which represented enhanced expression of primary transcript of pri-miR-23a in these cells. This increased Fluc activity decreased in differentiated P19 cells on day 3 and was further reduced on day 9. (B) Compared with CMV/Gluc, CMV/Gluc/3xPT_mir23 transfection revealed decreased Gluc activity, which represented higher mature miR-23a activities. This decrease of mature miR-23a activity was improved in differentiated P19 cells on day 3 and was further improved on day 9. There was a concordance between higher expression of pri-miR-23a and high activity of mature miR-23a in undifferentiated P19 cells and decreased, but-still-present, expression of pri-miR-23a (Fluc) and decreased repression (increase) of mature miR-23a (Gluc) in differentiated P19 cells during neurogenesis. Data are expressed as means (± SD) of triplicates. (C) RT-PCR of pri-miR-23a showed that expression of endogenous pri-miR-23a was reduced during P19 neurogenesis after RA treatment. β-Actin was used as control. (D) Real time qRT-PCR of mature miR-23a showed that endogenous mature miR-23a levels were reduced after P19 neurogenesis. U6 snRNA was used as control. Data are expressed as mean ± SD of triplicates. Ct = threshold cycle (Ct = CtmiRNA – CtU6RNA, Ct9d = Ct9d – Ct9d, Ct3d = Ct3d – Ct9d, Ctun = Ctun – Ct9d).

View larger version (31K): [in this window] [in a new window]   FIGURE 7.  In vivo visualization of expression of pri-miR-23a transcripts and activity of mature forms of miR-23a in nude mice. HeLa cells (A), 293 cells (B), and undifferentiated P19 cells (C) were transfected with miR23P639/Fluc and CMV/Gluc/3xPT_mir23 and were grafted (1 x 107 cells) to right side of mice. Same cells transfected with pGL3_basic and CMV/Gluc were grafted to left side as controls. Quantitative data from imaging in vivo were obtained by ROI analysis and are shown next to each small-animal picture. Gluc images were acquired first with coelenterazine, and then Fluc images were then acquired with D-luciferin. When viewing Fluc images we pay attention to increased bioluminescence on right side of grafted cells expressing pri-miR-23a transcripts. When viewing Gluc images we try to find decreased bioluminescence on right side compared with luciferase activity on left-sided CMV/Gluc controls, disclosing mature miR-23a activities. Fluc activities increased in all animals on right side, and Gluc activities decreased in HeLa and P19 cells but decreased the least in 293 cells transfected with CMV/Gluc/3xPT_mir23 on right side.