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MicroRNA-103a-3p controls proliferation and osteogenic differentiation of human adipose tissue-derived stromal cells.

Kim da S, Lee SY, Lee JH, Bae YC, Jung JS - Exp. Mol. Med. (2015)

Bottom Line: RNA interference-mediated downregulation of CDK6 and DICER1 in hADSCs inhibited their proliferation and osteogenic differentiation.The results of the current study indicate that miR-103a-3p regulates the osteogenic differentiation of hADSCs and proliferation of hADSCs by direct targeting of CDK6 and DICER1 partly.These findings further elucidate the molecular mechanisms governing the differentiation and proliferation of hADSCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, School of Medicine, Pusan National University, Yangsan, Korea.

ABSTRACT
The elucidation of the molecular mechanisms underlying the differentiation and proliferation of human adipose tissue-derived stromal cells (hADSCs) represents a critical step in the development of hADSCs-based cellular therapies. To examine the role of the microRNA-103a-3p (miR-103a-3p) in hADSCs functions, miR-103a-3p mimics were transfected into hADSCs in order to overexpress miR-103a-3p. Osteogenic differentiation was induced for 14 days in an osetogenic differentiation medium and assessed by using an Alizarin Red S stain. The regulation of the expression of CDK6 (cyclin-dependent kinase 6), a predicted target of miR-103a-3p, was determined by western blot, real-time PCR and luciferase reporter assays. Overexpression of miR-103a-3p inhibited the proliferation and osteogenic differentiation of hADSCs. In addition, it downregulated protein and mRNA levels of predicted target of miR-103a-3p (CDK6 and DICER1). In contrast, inhibition of miR-103a-3p with 2'O methyl antisense RNA increased the proliferation and osteogenic differentiation of hADSCs. The luciferase reporter activity of the construct containing the miR-103a-3p target site within the CDK6 and DICER1 3'-untranslated regions was lower in miR-103a-3p-transfected hADSCs than in control miRNA-transfected hADSCs. RNA interference-mediated downregulation of CDK6 and DICER1 in hADSCs inhibited their proliferation and osteogenic differentiation. The results of the current study indicate that miR-103a-3p regulates the osteogenic differentiation of hADSCs and proliferation of hADSCs by direct targeting of CDK6 and DICER1 partly. These findings further elucidate the molecular mechanisms governing the differentiation and proliferation of hADSCs.

No MeSH data available.


Inhibition of miR-103a-3p increases osteogenic differentiation and proliferation of hADSCs. (a) miR-103a-3p levels were determined in inhibitor control-(IH-miR-cont) or IH-miR-103a-3p–transfected hADSCs using real-time PCR. (b) hADSCs proliferation was determined by direct cell counting after oligonucleotide transfection. (c) Oligonucleotide-transfected hADSCs were grown for 10 days and, when the oligonucleotide-transfected hADSCs were grown to 80–90% confluence, osteogenic differentiation was induced for 2 weeks and determined by Alizarin Red S solution staining, which was quantified by absorbance at 562 nm. (d) Real-time PCR analysis of ALP in IH-miR-103a-3p-transfeted undifferentiated and differentiated cells. Total RNA was isolated at days after induction of differentiation. Internal control for expression analysis was GUSB. Data represent mean±s.e.m. (n=4). *P<0.05 compared with IH-miR-cont transfected hADSCs at 0 and 7 days. ALP, alkaline phosphatase; hADSC, human adipose tissue-derived stromal cell; miR, microRNA.
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fig2: Inhibition of miR-103a-3p increases osteogenic differentiation and proliferation of hADSCs. (a) miR-103a-3p levels were determined in inhibitor control-(IH-miR-cont) or IH-miR-103a-3p–transfected hADSCs using real-time PCR. (b) hADSCs proliferation was determined by direct cell counting after oligonucleotide transfection. (c) Oligonucleotide-transfected hADSCs were grown for 10 days and, when the oligonucleotide-transfected hADSCs were grown to 80–90% confluence, osteogenic differentiation was induced for 2 weeks and determined by Alizarin Red S solution staining, which was quantified by absorbance at 562 nm. (d) Real-time PCR analysis of ALP in IH-miR-103a-3p-transfeted undifferentiated and differentiated cells. Total RNA was isolated at days after induction of differentiation. Internal control for expression analysis was GUSB. Data represent mean±s.e.m. (n=4). *P<0.05 compared with IH-miR-cont transfected hADSCs at 0 and 7 days. ALP, alkaline phosphatase; hADSC, human adipose tissue-derived stromal cell; miR, microRNA.

Mentions: To investigate the effect of miR-103a-3p inhibition on hADSCs differentiation, hADSCs were transfected with a specific miRNA inhibitor (IH-miR-103a-3p). Real-time PCR analysis showed that transfection of IH-miR-103a-3p effectively downregulated miR-103a-3p expression in hADSCs (Figure 2a). The cell counting experiment showed that IH-miR-103a-3p enhanced hADSCs proliferation compared with control oligonucleotide-transfected cells (Figure 2b).


MicroRNA-103a-3p controls proliferation and osteogenic differentiation of human adipose tissue-derived stromal cells.

Kim da S, Lee SY, Lee JH, Bae YC, Jung JS - Exp. Mol. Med. (2015)

Inhibition of miR-103a-3p increases osteogenic differentiation and proliferation of hADSCs. (a) miR-103a-3p levels were determined in inhibitor control-(IH-miR-cont) or IH-miR-103a-3p–transfected hADSCs using real-time PCR. (b) hADSCs proliferation was determined by direct cell counting after oligonucleotide transfection. (c) Oligonucleotide-transfected hADSCs were grown for 10 days and, when the oligonucleotide-transfected hADSCs were grown to 80–90% confluence, osteogenic differentiation was induced for 2 weeks and determined by Alizarin Red S solution staining, which was quantified by absorbance at 562 nm. (d) Real-time PCR analysis of ALP in IH-miR-103a-3p-transfeted undifferentiated and differentiated cells. Total RNA was isolated at days after induction of differentiation. Internal control for expression analysis was GUSB. Data represent mean±s.e.m. (n=4). *P<0.05 compared with IH-miR-cont transfected hADSCs at 0 and 7 days. ALP, alkaline phosphatase; hADSC, human adipose tissue-derived stromal cell; miR, microRNA.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig2: Inhibition of miR-103a-3p increases osteogenic differentiation and proliferation of hADSCs. (a) miR-103a-3p levels were determined in inhibitor control-(IH-miR-cont) or IH-miR-103a-3p–transfected hADSCs using real-time PCR. (b) hADSCs proliferation was determined by direct cell counting after oligonucleotide transfection. (c) Oligonucleotide-transfected hADSCs were grown for 10 days and, when the oligonucleotide-transfected hADSCs were grown to 80–90% confluence, osteogenic differentiation was induced for 2 weeks and determined by Alizarin Red S solution staining, which was quantified by absorbance at 562 nm. (d) Real-time PCR analysis of ALP in IH-miR-103a-3p-transfeted undifferentiated and differentiated cells. Total RNA was isolated at days after induction of differentiation. Internal control for expression analysis was GUSB. Data represent mean±s.e.m. (n=4). *P<0.05 compared with IH-miR-cont transfected hADSCs at 0 and 7 days. ALP, alkaline phosphatase; hADSC, human adipose tissue-derived stromal cell; miR, microRNA.
Mentions: To investigate the effect of miR-103a-3p inhibition on hADSCs differentiation, hADSCs were transfected with a specific miRNA inhibitor (IH-miR-103a-3p). Real-time PCR analysis showed that transfection of IH-miR-103a-3p effectively downregulated miR-103a-3p expression in hADSCs (Figure 2a). The cell counting experiment showed that IH-miR-103a-3p enhanced hADSCs proliferation compared with control oligonucleotide-transfected cells (Figure 2b).

Bottom Line: RNA interference-mediated downregulation of CDK6 and DICER1 in hADSCs inhibited their proliferation and osteogenic differentiation.The results of the current study indicate that miR-103a-3p regulates the osteogenic differentiation of hADSCs and proliferation of hADSCs by direct targeting of CDK6 and DICER1 partly.These findings further elucidate the molecular mechanisms governing the differentiation and proliferation of hADSCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, School of Medicine, Pusan National University, Yangsan, Korea.

ABSTRACT
The elucidation of the molecular mechanisms underlying the differentiation and proliferation of human adipose tissue-derived stromal cells (hADSCs) represents a critical step in the development of hADSCs-based cellular therapies. To examine the role of the microRNA-103a-3p (miR-103a-3p) in hADSCs functions, miR-103a-3p mimics were transfected into hADSCs in order to overexpress miR-103a-3p. Osteogenic differentiation was induced for 14 days in an osetogenic differentiation medium and assessed by using an Alizarin Red S stain. The regulation of the expression of CDK6 (cyclin-dependent kinase 6), a predicted target of miR-103a-3p, was determined by western blot, real-time PCR and luciferase reporter assays. Overexpression of miR-103a-3p inhibited the proliferation and osteogenic differentiation of hADSCs. In addition, it downregulated protein and mRNA levels of predicted target of miR-103a-3p (CDK6 and DICER1). In contrast, inhibition of miR-103a-3p with 2'O methyl antisense RNA increased the proliferation and osteogenic differentiation of hADSCs. The luciferase reporter activity of the construct containing the miR-103a-3p target site within the CDK6 and DICER1 3'-untranslated regions was lower in miR-103a-3p-transfected hADSCs than in control miRNA-transfected hADSCs. RNA interference-mediated downregulation of CDK6 and DICER1 in hADSCs inhibited their proliferation and osteogenic differentiation. The results of the current study indicate that miR-103a-3p regulates the osteogenic differentiation of hADSCs and proliferation of hADSCs by direct targeting of CDK6 and DICER1 partly. These findings further elucidate the molecular mechanisms governing the differentiation and proliferation of hADSCs.

No MeSH data available.