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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.


CDK6 siRNA inhibits osteogenic differentiation and proliferation of hADSCs. (a) CDK6 mRNA levels were determined in control-(si-cont) or CDK6 oligonucleotide (si-CDK6) transfected hADSCs by using real-time PCR. Internal control for expression analysis was GUSB. (b) hADSCs proliferation was determined by direct cell counting. (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 staining, which was quantified by absorbance at 562 nm. (d) Real-time PCR analysis of ALP, and Runx2 in si-CDK6-transfected undifferentiated cells. Internal control for expression analysis was GUSB. Data represent mean±s.e.m. (n=4). *P<0.05 compared with si-cont-transfected hADSCs. ALP, alkaline phosphatase; hADSC, human adipose tissue-derived stromal cell; miR, microRNA; si-CDK6, CDK6 small interfering RNA.
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fig4: CDK6 siRNA inhibits osteogenic differentiation and proliferation of hADSCs. (a) CDK6 mRNA levels were determined in control-(si-cont) or CDK6 oligonucleotide (si-CDK6) transfected hADSCs by using real-time PCR. Internal control for expression analysis was GUSB. (b) hADSCs proliferation was determined by direct cell counting. (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 staining, which was quantified by absorbance at 562 nm. (d) Real-time PCR analysis of ALP, and Runx2 in si-CDK6-transfected undifferentiated cells. Internal control for expression analysis was GUSB. Data represent mean±s.e.m. (n=4). *P<0.05 compared with si-cont-transfected hADSCs. ALP, alkaline phosphatase; hADSC, human adipose tissue-derived stromal cell; miR, microRNA; si-CDK6, CDK6 small interfering RNA.

Mentions: To determine the role of CDK6 in osteogenic differentiation and proliferation of hADSCs, CDK6 expression was suppressed in hADSCs with an RNA interference technique using CDK6 siRNA (CDK6 siRNA) transfection. Real-time PCR analysis confirmed that CDK6 siRNA effectively inhibited CDK6 expression in hADSCs (Figure 4a). Direct cell counting showed that CDK6 siRNA-transfected hADSCs proliferated less than the control cells (Figure 4b).


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)

CDK6 siRNA inhibits osteogenic differentiation and proliferation of hADSCs. (a) CDK6 mRNA levels were determined in control-(si-cont) or CDK6 oligonucleotide (si-CDK6) transfected hADSCs by using real-time PCR. Internal control for expression analysis was GUSB. (b) hADSCs proliferation was determined by direct cell counting. (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 staining, which was quantified by absorbance at 562 nm. (d) Real-time PCR analysis of ALP, and Runx2 in si-CDK6-transfected undifferentiated cells. Internal control for expression analysis was GUSB. Data represent mean±s.e.m. (n=4). *P<0.05 compared with si-cont-transfected hADSCs. ALP, alkaline phosphatase; hADSC, human adipose tissue-derived stromal cell; miR, microRNA; si-CDK6, CDK6 small interfering RNA.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4525297&req=5

fig4: CDK6 siRNA inhibits osteogenic differentiation and proliferation of hADSCs. (a) CDK6 mRNA levels were determined in control-(si-cont) or CDK6 oligonucleotide (si-CDK6) transfected hADSCs by using real-time PCR. Internal control for expression analysis was GUSB. (b) hADSCs proliferation was determined by direct cell counting. (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 staining, which was quantified by absorbance at 562 nm. (d) Real-time PCR analysis of ALP, and Runx2 in si-CDK6-transfected undifferentiated cells. Internal control for expression analysis was GUSB. Data represent mean±s.e.m. (n=4). *P<0.05 compared with si-cont-transfected hADSCs. ALP, alkaline phosphatase; hADSC, human adipose tissue-derived stromal cell; miR, microRNA; si-CDK6, CDK6 small interfering RNA.
Mentions: To determine the role of CDK6 in osteogenic differentiation and proliferation of hADSCs, CDK6 expression was suppressed in hADSCs with an RNA interference technique using CDK6 siRNA (CDK6 siRNA) transfection. Real-time PCR analysis confirmed that CDK6 siRNA effectively inhibited CDK6 expression in hADSCs (Figure 4a). Direct cell counting showed that CDK6 siRNA-transfected hADSCs proliferated less than the control cells (Figure 4b).

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.