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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.


Overexpression of miR-103a-3p inhibits osteogenic differentiation and proliferation of hADSCs. (a) miR-103a-3p levels were determined in mimic control (mimic miR-cont) or mimic 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 2 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, which was quantified by absorbance at 562 nm. (d) Real-time PCR analysis of ALP, in mimic miR-103a-3p-transfected 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 mimic 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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fig1: Overexpression of miR-103a-3p inhibits osteogenic differentiation and proliferation of hADSCs. (a) miR-103a-3p levels were determined in mimic control (mimic miR-cont) or mimic 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 2 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, which was quantified by absorbance at 562 nm. (d) Real-time PCR analysis of ALP, in mimic miR-103a-3p-transfected 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 mimic miR-cont-transfected hADSCs at 0 and 7 days. ALP, alkaline phosphatase; hADSC, human adipose tissue-derived stromal cell; miR, microRNA.

Mentions: The miRNAs that showed high levels of expression in a preliminary miRNA microarray study were chosen and then differentiation-related molecules were targeted by using the miRWalk database.23 To examine the role of miR-103a-3p in hADSCs functions, miR-103a-3p mimics were transfected into hADSCs in order to overexpress miR-103a-3p. miRNA mimics augment the function of endogenous miRNAs for easier detection of phenotypic changes. Real-time PCR analysis showed that miR-103a-3p-transfected hADSCs had increased levels of miR-103a-3p expression (Figure 1a).


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)

Overexpression of miR-103a-3p inhibits osteogenic differentiation and proliferation of hADSCs. (a) miR-103a-3p levels were determined in mimic control (mimic miR-cont) or mimic 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 2 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, which was quantified by absorbance at 562 nm. (d) Real-time PCR analysis of ALP, in mimic miR-103a-3p-transfected 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 mimic 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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getmorefigures.php?uid=PMC4525297&req=5

fig1: Overexpression of miR-103a-3p inhibits osteogenic differentiation and proliferation of hADSCs. (a) miR-103a-3p levels were determined in mimic control (mimic miR-cont) or mimic 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 2 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, which was quantified by absorbance at 562 nm. (d) Real-time PCR analysis of ALP, in mimic miR-103a-3p-transfected 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 mimic miR-cont-transfected hADSCs at 0 and 7 days. ALP, alkaline phosphatase; hADSC, human adipose tissue-derived stromal cell; miR, microRNA.
Mentions: The miRNAs that showed high levels of expression in a preliminary miRNA microarray study were chosen and then differentiation-related molecules were targeted by using the miRWalk database.23 To examine the role of miR-103a-3p in hADSCs functions, miR-103a-3p mimics were transfected into hADSCs in order to overexpress miR-103a-3p. miRNA mimics augment the function of endogenous miRNAs for easier detection of phenotypic changes. Real-time PCR analysis showed that miR-103a-3p-transfected hADSCs had increased levels of miR-103a-3p expression (Figure 1a).

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.