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Bioimaging of microRNA124a-independent neuronal differentiation of human G2 neural stem cells.

Lee J, Hwang do W, Kim SU, Lee DS, Lee YS, Heo H, Ali BA, Al-Khedhairy AA, Kim S - FEBS Open Bio (2015)

Bottom Line: Our recently developed miRNA reporter imaging vectors (CMV/Gluc/3×PT_miR-9 and CMV/Gluc/3×PT_miR-124a) containing Gaussia luciferase, CMV promoter and three copies of complementary nucleotides of each corresponding miRNA showed that luciferase activity from CMV/Gluc/3×PT_miR-9 was gradually decreased both in vitro and in vivo in G2 cells induced to differentiate into neurons.However, in vitro and in vivo bioluminescence signals for CMV/Gluc/3×PT_miR-124a were not significantly different between undifferentiated and differentiated G2 cells.Our results demonstrate that biogenesis of neuron-specific miR-124a is not necessary for doxycycline-dependent neurogenesis of G2 cells.

View Article: PubMed Central - PubMed

Affiliation: Institute for Bio-Medical Convergence, College of Medicine, Catholic Kwandong University, Gangneung-si, Gangwon-do 270-701, Republic of Korea ; Catholic Kwandong University International St. Mary's Hospital, Incheon Metropolitan City 404-834, Republic of Korea.

ABSTRACT
Evaluation of the function of microRNAs (miRNAs or miRs) through miRNA expression profiles during neuronal differentiation plays a critical role not only in identifying unique miRNAs relevant to cellular development but also in understanding regulatory functions of the cell-specific miRNAs in living organisms. Here, we examined the microarray-based miRNA expression profiles of G2 cells (recently developed human neural stem cells) and monitored the expression pattern of known neuron-specific miR-9 and miR-124a during neuronal differentiation of G2 cells in vitro and in vivo. Of 500 miRNAs analyzed by microarray of G2 cells, the expression of 90 miRNAs was significantly increased during doxycycline-dependent neuronal differentiation of G2 cells and about 60 miRNAs showed a gradual enhancement of gene expression as neuronal differentiation progressed. Real-time PCR showed that expression of endogenous mature miR-9 was continuously and gradually increased in a pattern dependent on the period of neuronal differentiation of G2 cells while the increased expression of neuron-specific mature miR-124a was barely observed during neurogenesis. Our recently developed miRNA reporter imaging vectors (CMV/Gluc/3×PT_miR-9 and CMV/Gluc/3×PT_miR-124a) containing Gaussia luciferase, CMV promoter and three copies of complementary nucleotides of each corresponding miRNA showed that luciferase activity from CMV/Gluc/3×PT_miR-9 was gradually decreased both in vitro and in vivo in G2 cells induced to differentiate into neurons. However, in vitro and in vivo bioluminescence signals for CMV/Gluc/3×PT_miR-124a were not significantly different between undifferentiated and differentiated G2 cells. Our results demonstrate that biogenesis of neuron-specific miR-124a is not necessary for doxycycline-dependent neurogenesis of G2 cells.

No MeSH data available.


Related in: MedlinePlus

In vitro validation of miR-9 and miR-124a expression patterns in neuronal differentiation of G2 cells. (A) Small RNA was extracted from G2 cells at 0, 2, 4, and 6 days after neuronal induction by the withdrawal of doxycycline. Quantitative RT-PCR analysis showed that the endogenous mature miR-9 expression level was gradually increased following the induction of neuronal differentiation of G2 cells. In contrast, there was no significant change in the expression level of mature miR-124a during neurogenesis. U6 snRNA was used as an internal control. Results are expressed as the mean (SD) of triplicate experiments. (B) qRT-PCR analysis was performed to assess the expression level of an miR-124a target gene using specific primers for VAMP3, a putative target gene for miR-124a. Similar expression patterns of VAMP3 were detected at each neuronal differentiation time point in G2 cells. (C) The constructed CMV/Gluc/3×PT_miR-9 (or CMV/Gluc/3×PT_miR-124a) plasmid vector was transiently transfected into undifferentiated G2 cells, and Gaussia luciferase activity was measured at each neuronal differentiation time point in G2 cells. A gradual decrease in the level of Gaussia luciferase was found in the treatment group of CMV/Gluc/3×PT_miR-9, due to the increase in expression of miRNA-9 during neuronal differentiation of G2 cells. CMV/Gluc/3×PT_miR-124a-treated G2 cells showed similar activity levels of Gaussia luciferase until 2 days after neuronal differentiation. (*P < 0.05, **P < 0.01).
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f0015: In vitro validation of miR-9 and miR-124a expression patterns in neuronal differentiation of G2 cells. (A) Small RNA was extracted from G2 cells at 0, 2, 4, and 6 days after neuronal induction by the withdrawal of doxycycline. Quantitative RT-PCR analysis showed that the endogenous mature miR-9 expression level was gradually increased following the induction of neuronal differentiation of G2 cells. In contrast, there was no significant change in the expression level of mature miR-124a during neurogenesis. U6 snRNA was used as an internal control. Results are expressed as the mean (SD) of triplicate experiments. (B) qRT-PCR analysis was performed to assess the expression level of an miR-124a target gene using specific primers for VAMP3, a putative target gene for miR-124a. Similar expression patterns of VAMP3 were detected at each neuronal differentiation time point in G2 cells. (C) The constructed CMV/Gluc/3×PT_miR-9 (or CMV/Gluc/3×PT_miR-124a) plasmid vector was transiently transfected into undifferentiated G2 cells, and Gaussia luciferase activity was measured at each neuronal differentiation time point in G2 cells. A gradual decrease in the level of Gaussia luciferase was found in the treatment group of CMV/Gluc/3×PT_miR-9, due to the increase in expression of miRNA-9 during neuronal differentiation of G2 cells. CMV/Gluc/3×PT_miR-124a-treated G2 cells showed similar activity levels of Gaussia luciferase until 2 days after neuronal differentiation. (*P < 0.05, **P < 0.01).

Mentions: Among the miRNAs aberrantly expressed during neurogenesis of G2 cells, miR-124a was selected to use for verification of its expression using real-time PCR. In normal neurogenesis, miR-124a gradually increases in expression during neurogenesis and is highly neuron-specific [6,19–21]. Similarly to the results of the microRNA microarray, real-time PCR measurement of miR-124a showed no significant gradual increase in miRNA expression after differentiation of G2 cells while expression of miR-9, used as a positive control, was gradually increased over time during neuronal differentiation of G2 cells (Fig. 3A). The miR-124a expression pattern was also studied in P19 cells, which are embryonal teratocarcinoma cells that have been differentiated into a neuronal lineage controlled by treatment with RA. Similar to results shown in other reports for the miR-124a expression pattern during neurogenesis, the miR-124a expression level was gradually increased during the progression of neuronal differentiation in RA-treated P19 cells [7,19] (Supplementary Fig. 1).


Bioimaging of microRNA124a-independent neuronal differentiation of human G2 neural stem cells.

Lee J, Hwang do W, Kim SU, Lee DS, Lee YS, Heo H, Ali BA, Al-Khedhairy AA, Kim S - FEBS Open Bio (2015)

In vitro validation of miR-9 and miR-124a expression patterns in neuronal differentiation of G2 cells. (A) Small RNA was extracted from G2 cells at 0, 2, 4, and 6 days after neuronal induction by the withdrawal of doxycycline. Quantitative RT-PCR analysis showed that the endogenous mature miR-9 expression level was gradually increased following the induction of neuronal differentiation of G2 cells. In contrast, there was no significant change in the expression level of mature miR-124a during neurogenesis. U6 snRNA was used as an internal control. Results are expressed as the mean (SD) of triplicate experiments. (B) qRT-PCR analysis was performed to assess the expression level of an miR-124a target gene using specific primers for VAMP3, a putative target gene for miR-124a. Similar expression patterns of VAMP3 were detected at each neuronal differentiation time point in G2 cells. (C) The constructed CMV/Gluc/3×PT_miR-9 (or CMV/Gluc/3×PT_miR-124a) plasmid vector was transiently transfected into undifferentiated G2 cells, and Gaussia luciferase activity was measured at each neuronal differentiation time point in G2 cells. A gradual decrease in the level of Gaussia luciferase was found in the treatment group of CMV/Gluc/3×PT_miR-9, due to the increase in expression of miRNA-9 during neuronal differentiation of G2 cells. CMV/Gluc/3×PT_miR-124a-treated G2 cells showed similar activity levels of Gaussia luciferase until 2 days after neuronal differentiation. (*P < 0.05, **P < 0.01).
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f0015: In vitro validation of miR-9 and miR-124a expression patterns in neuronal differentiation of G2 cells. (A) Small RNA was extracted from G2 cells at 0, 2, 4, and 6 days after neuronal induction by the withdrawal of doxycycline. Quantitative RT-PCR analysis showed that the endogenous mature miR-9 expression level was gradually increased following the induction of neuronal differentiation of G2 cells. In contrast, there was no significant change in the expression level of mature miR-124a during neurogenesis. U6 snRNA was used as an internal control. Results are expressed as the mean (SD) of triplicate experiments. (B) qRT-PCR analysis was performed to assess the expression level of an miR-124a target gene using specific primers for VAMP3, a putative target gene for miR-124a. Similar expression patterns of VAMP3 were detected at each neuronal differentiation time point in G2 cells. (C) The constructed CMV/Gluc/3×PT_miR-9 (or CMV/Gluc/3×PT_miR-124a) plasmid vector was transiently transfected into undifferentiated G2 cells, and Gaussia luciferase activity was measured at each neuronal differentiation time point in G2 cells. A gradual decrease in the level of Gaussia luciferase was found in the treatment group of CMV/Gluc/3×PT_miR-9, due to the increase in expression of miRNA-9 during neuronal differentiation of G2 cells. CMV/Gluc/3×PT_miR-124a-treated G2 cells showed similar activity levels of Gaussia luciferase until 2 days after neuronal differentiation. (*P < 0.05, **P < 0.01).
Mentions: Among the miRNAs aberrantly expressed during neurogenesis of G2 cells, miR-124a was selected to use for verification of its expression using real-time PCR. In normal neurogenesis, miR-124a gradually increases in expression during neurogenesis and is highly neuron-specific [6,19–21]. Similarly to the results of the microRNA microarray, real-time PCR measurement of miR-124a showed no significant gradual increase in miRNA expression after differentiation of G2 cells while expression of miR-9, used as a positive control, was gradually increased over time during neuronal differentiation of G2 cells (Fig. 3A). The miR-124a expression pattern was also studied in P19 cells, which are embryonal teratocarcinoma cells that have been differentiated into a neuronal lineage controlled by treatment with RA. Similar to results shown in other reports for the miR-124a expression pattern during neurogenesis, the miR-124a expression level was gradually increased during the progression of neuronal differentiation in RA-treated P19 cells [7,19] (Supplementary Fig. 1).

Bottom Line: Our recently developed miRNA reporter imaging vectors (CMV/Gluc/3×PT_miR-9 and CMV/Gluc/3×PT_miR-124a) containing Gaussia luciferase, CMV promoter and three copies of complementary nucleotides of each corresponding miRNA showed that luciferase activity from CMV/Gluc/3×PT_miR-9 was gradually decreased both in vitro and in vivo in G2 cells induced to differentiate into neurons.However, in vitro and in vivo bioluminescence signals for CMV/Gluc/3×PT_miR-124a were not significantly different between undifferentiated and differentiated G2 cells.Our results demonstrate that biogenesis of neuron-specific miR-124a is not necessary for doxycycline-dependent neurogenesis of G2 cells.

View Article: PubMed Central - PubMed

Affiliation: Institute for Bio-Medical Convergence, College of Medicine, Catholic Kwandong University, Gangneung-si, Gangwon-do 270-701, Republic of Korea ; Catholic Kwandong University International St. Mary's Hospital, Incheon Metropolitan City 404-834, Republic of Korea.

ABSTRACT
Evaluation of the function of microRNAs (miRNAs or miRs) through miRNA expression profiles during neuronal differentiation plays a critical role not only in identifying unique miRNAs relevant to cellular development but also in understanding regulatory functions of the cell-specific miRNAs in living organisms. Here, we examined the microarray-based miRNA expression profiles of G2 cells (recently developed human neural stem cells) and monitored the expression pattern of known neuron-specific miR-9 and miR-124a during neuronal differentiation of G2 cells in vitro and in vivo. Of 500 miRNAs analyzed by microarray of G2 cells, the expression of 90 miRNAs was significantly increased during doxycycline-dependent neuronal differentiation of G2 cells and about 60 miRNAs showed a gradual enhancement of gene expression as neuronal differentiation progressed. Real-time PCR showed that expression of endogenous mature miR-9 was continuously and gradually increased in a pattern dependent on the period of neuronal differentiation of G2 cells while the increased expression of neuron-specific mature miR-124a was barely observed during neurogenesis. Our recently developed miRNA reporter imaging vectors (CMV/Gluc/3×PT_miR-9 and CMV/Gluc/3×PT_miR-124a) containing Gaussia luciferase, CMV promoter and three copies of complementary nucleotides of each corresponding miRNA showed that luciferase activity from CMV/Gluc/3×PT_miR-9 was gradually decreased both in vitro and in vivo in G2 cells induced to differentiate into neurons. However, in vitro and in vivo bioluminescence signals for CMV/Gluc/3×PT_miR-124a were not significantly different between undifferentiated and differentiated G2 cells. Our results demonstrate that biogenesis of neuron-specific miR-124a is not necessary for doxycycline-dependent neurogenesis of G2 cells.

No MeSH data available.


Related in: MedlinePlus