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A feedback regulatory loop involving microRNA-9 and nuclear receptor TLX in neural stem cell fate determination.

Zhao C, Sun G, Li S, Shi Y - Nat. Struct. Mol. Biol. (2009)

Bottom Line: Here we show that miR-9 suppresses TLX expression to negatively regulate neural stem cell proliferation and accelerate neural differentiation.Moreover, TLX represses expression of the miR-9 pri-miRNA.By forming a negative regulatory loop with TLX, miR-9 provides a model for controlling the balance between neural stem cell proliferation and differentiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosciences, Center for Gene Expression and Drug Discovery, Beckman Research Institute of City of Hope, Duarte, California, USA.

ABSTRACT
MicroRNAs have been implicated as having important roles in stem cell biology. MicroRNA-9 (miR-9) is expressed specifically in neurogenic areas of the brain and may be involved in neural stem cell self-renewal and differentiation. We showed previously that the nuclear receptor TLX is an essential regulator of neural stem cell self-renewal. Here we show that miR-9 suppresses TLX expression to negatively regulate neural stem cell proliferation and accelerate neural differentiation. Introducing a TLX expression vector that is not prone to miR-9 regulation rescued miR-9-induced proliferation deficiency and inhibited precocious differentiation. In utero electroporation of miR-9 in embryonic brains led to premature differentiation and outward migration of the transfected neural stem cells. Moreover, TLX represses expression of the miR-9 pri-miRNA. By forming a negative regulatory loop with TLX, miR-9 provides a model for controlling the balance between neural stem cell proliferation and differentiation.

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miR-9 antisense RNA promotes neural stem cell proliferationa. 2′-O-methyl miR-9 antisense RNA knocks down miR-9 mature form analyzed by Northern blot analysis. 2′-O-methyl antisense GFP RNA was included as a negative control (C) in all sections. U6 was included as a loading control. b. Expression of TLX and p21 in 2′-O-methyl miR-9 antisense RNA-treated neural stem cells analyzed by RT-PCR. GAPDH was included as a loading control. c. Neural stem cells were transfected with control RNA and 2′-O-methyl miR-9 antisense RNA. The transfected cells were labeled by BrdU staining (green). Merged panels show BrdU staining along with phase contrast images. d. Quantification of BrdU+ cells in control (C) and 2′-O-methyl miR-9 antisense RNA-treated neural stem cells. s.d. is represented by error bars. * p=0.03 by Student's t-test.
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Figure 4: miR-9 antisense RNA promotes neural stem cell proliferationa. 2′-O-methyl miR-9 antisense RNA knocks down miR-9 mature form analyzed by Northern blot analysis. 2′-O-methyl antisense GFP RNA was included as a negative control (C) in all sections. U6 was included as a loading control. b. Expression of TLX and p21 in 2′-O-methyl miR-9 antisense RNA-treated neural stem cells analyzed by RT-PCR. GAPDH was included as a loading control. c. Neural stem cells were transfected with control RNA and 2′-O-methyl miR-9 antisense RNA. The transfected cells were labeled by BrdU staining (green). Merged panels show BrdU staining along with phase contrast images. d. Quantification of BrdU+ cells in control (C) and 2′-O-methyl miR-9 antisense RNA-treated neural stem cells. s.d. is represented by error bars. * p=0.03 by Student's t-test.

Mentions: Using 2′-O-methyl antisense RNA oligonucleotides as small RNA inhibitors28,29, the role of miR-9 in neural stem cell proliferation was further investigated. 2′-O-methyl antisense oligonucleotide against miR-9 was synthesized and transfected into neural stem cells with 2′-O-methyl antisense oligonucleotide against green fluorescent protein (GFP) included as a negative control. Treatment of antisense oligonucleotides against miR-9 led to substantial knockdown of miR-9 mature forms (Fig. 4a). The expression of TLX was upregulated in miR-9 antisense RNA-treated neural stem cells, along with decreased expression of p21 (Fig. 4b). BrdU labeling analysis revealed that knockdown of miR-9 led to an increase in cell proliferation (1.37-fold, Fig. 4c, d), consistent with enhanced cell proliferation in TLXΔ3′ UTR-transduced neural stem cells (Fig. 3b, c).


A feedback regulatory loop involving microRNA-9 and nuclear receptor TLX in neural stem cell fate determination.

Zhao C, Sun G, Li S, Shi Y - Nat. Struct. Mol. Biol. (2009)

miR-9 antisense RNA promotes neural stem cell proliferationa. 2′-O-methyl miR-9 antisense RNA knocks down miR-9 mature form analyzed by Northern blot analysis. 2′-O-methyl antisense GFP RNA was included as a negative control (C) in all sections. U6 was included as a loading control. b. Expression of TLX and p21 in 2′-O-methyl miR-9 antisense RNA-treated neural stem cells analyzed by RT-PCR. GAPDH was included as a loading control. c. Neural stem cells were transfected with control RNA and 2′-O-methyl miR-9 antisense RNA. The transfected cells were labeled by BrdU staining (green). Merged panels show BrdU staining along with phase contrast images. d. Quantification of BrdU+ cells in control (C) and 2′-O-methyl miR-9 antisense RNA-treated neural stem cells. s.d. is represented by error bars. * p=0.03 by Student's t-test.
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Related In: Results  -  Collection

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Figure 4: miR-9 antisense RNA promotes neural stem cell proliferationa. 2′-O-methyl miR-9 antisense RNA knocks down miR-9 mature form analyzed by Northern blot analysis. 2′-O-methyl antisense GFP RNA was included as a negative control (C) in all sections. U6 was included as a loading control. b. Expression of TLX and p21 in 2′-O-methyl miR-9 antisense RNA-treated neural stem cells analyzed by RT-PCR. GAPDH was included as a loading control. c. Neural stem cells were transfected with control RNA and 2′-O-methyl miR-9 antisense RNA. The transfected cells were labeled by BrdU staining (green). Merged panels show BrdU staining along with phase contrast images. d. Quantification of BrdU+ cells in control (C) and 2′-O-methyl miR-9 antisense RNA-treated neural stem cells. s.d. is represented by error bars. * p=0.03 by Student's t-test.
Mentions: Using 2′-O-methyl antisense RNA oligonucleotides as small RNA inhibitors28,29, the role of miR-9 in neural stem cell proliferation was further investigated. 2′-O-methyl antisense oligonucleotide against miR-9 was synthesized and transfected into neural stem cells with 2′-O-methyl antisense oligonucleotide against green fluorescent protein (GFP) included as a negative control. Treatment of antisense oligonucleotides against miR-9 led to substantial knockdown of miR-9 mature forms (Fig. 4a). The expression of TLX was upregulated in miR-9 antisense RNA-treated neural stem cells, along with decreased expression of p21 (Fig. 4b). BrdU labeling analysis revealed that knockdown of miR-9 led to an increase in cell proliferation (1.37-fold, Fig. 4c, d), consistent with enhanced cell proliferation in TLXΔ3′ UTR-transduced neural stem cells (Fig. 3b, c).

Bottom Line: Here we show that miR-9 suppresses TLX expression to negatively regulate neural stem cell proliferation and accelerate neural differentiation.Moreover, TLX represses expression of the miR-9 pri-miRNA.By forming a negative regulatory loop with TLX, miR-9 provides a model for controlling the balance between neural stem cell proliferation and differentiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosciences, Center for Gene Expression and Drug Discovery, Beckman Research Institute of City of Hope, Duarte, California, USA.

ABSTRACT
MicroRNAs have been implicated as having important roles in stem cell biology. MicroRNA-9 (miR-9) is expressed specifically in neurogenic areas of the brain and may be involved in neural stem cell self-renewal and differentiation. We showed previously that the nuclear receptor TLX is an essential regulator of neural stem cell self-renewal. Here we show that miR-9 suppresses TLX expression to negatively regulate neural stem cell proliferation and accelerate neural differentiation. Introducing a TLX expression vector that is not prone to miR-9 regulation rescued miR-9-induced proliferation deficiency and inhibited precocious differentiation. In utero electroporation of miR-9 in embryonic brains led to premature differentiation and outward migration of the transfected neural stem cells. Moreover, TLX represses expression of the miR-9 pri-miRNA. By forming a negative regulatory loop with TLX, miR-9 provides a model for controlling the balance between neural stem cell proliferation and differentiation.

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