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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.


DICER1 RNAi inhibits osteogenic differentiation and proliferation of hADSCs. (a) DICER1 mRNA levels were determined in control-(si-cont) or DICER1 oligonucleotide-(si-DICER1) transfected hADSCs by using real-time PCR. (b) hADSCs proliferation was determined by direct cell counting. (c) Osteogenic differentiation were determined same as Figure 1c method. Data represent mean±s.e.m. (n=4). *P<0.05 compared with si-con-transfected hADSCs. hADSC, human adipose tissue-derived stromal cell; si-DICER1, DICER1 small interfering RNA.
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fig6: DICER1 RNAi inhibits osteogenic differentiation and proliferation of hADSCs. (a) DICER1 mRNA levels were determined in control-(si-cont) or DICER1 oligonucleotide-(si-DICER1) transfected hADSCs by using real-time PCR. (b) hADSCs proliferation was determined by direct cell counting. (c) Osteogenic differentiation were determined same as Figure 1c method. Data represent mean±s.e.m. (n=4). *P<0.05 compared with si-con-transfected hADSCs. hADSC, human adipose tissue-derived stromal cell; si-DICER1, DICER1 small interfering RNA.

Mentions: To determine the role of DICER1 in osteogenic differentiation and proliferation of hADSCs, DICER1 expression was suppressed in hADSCs with an RNA interference technique using DICER1 siRNA (DICER1 siRNA) transfection. Real-time PCR analysis confirmed that DICER1 siRNA effectively inhibited DICER1 expression in hADSCs (Figure 6a). The effect of DICER1 downregulation on hADSCs proliferation was also determined. Direct cell counting showed that DICER1 siRNA-transfected hADSCs proliferated less than the control cells (Figure 6b).


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)

DICER1 RNAi inhibits osteogenic differentiation and proliferation of hADSCs. (a) DICER1 mRNA levels were determined in control-(si-cont) or DICER1 oligonucleotide-(si-DICER1) transfected hADSCs by using real-time PCR. (b) hADSCs proliferation was determined by direct cell counting. (c) Osteogenic differentiation were determined same as Figure 1c method. Data represent mean±s.e.m. (n=4). *P<0.05 compared with si-con-transfected hADSCs. hADSC, human adipose tissue-derived stromal cell; si-DICER1, DICER1 small interfering RNA.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig6: DICER1 RNAi inhibits osteogenic differentiation and proliferation of hADSCs. (a) DICER1 mRNA levels were determined in control-(si-cont) or DICER1 oligonucleotide-(si-DICER1) transfected hADSCs by using real-time PCR. (b) hADSCs proliferation was determined by direct cell counting. (c) Osteogenic differentiation were determined same as Figure 1c method. Data represent mean±s.e.m. (n=4). *P<0.05 compared with si-con-transfected hADSCs. hADSC, human adipose tissue-derived stromal cell; si-DICER1, DICER1 small interfering RNA.
Mentions: To determine the role of DICER1 in osteogenic differentiation and proliferation of hADSCs, DICER1 expression was suppressed in hADSCs with an RNA interference technique using DICER1 siRNA (DICER1 siRNA) transfection. Real-time PCR analysis confirmed that DICER1 siRNA effectively inhibited DICER1 expression in hADSCs (Figure 6a). The effect of DICER1 downregulation on hADSCs proliferation was also determined. Direct cell counting showed that DICER1 siRNA-transfected hADSCs proliferated less than the control cells (Figure 6b).

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.