J Vet Sci.  2013 Mar;14(1):69-76. 10.4142/jvs.2013.14.1.69.

Survival of hypoxic human mesenchymal stem cells is enhanced by a positive feedback loop involving miR-210 and hypoxia-inducible factor 1

• Chang W
• Lee CY
• Park JH
• Park MS
• Maeng LS
• Yoon CS
• Lee MY
• Hwang KC
• Chung YA

• KMID: 1482815
• DOI: http://doi.org/10.4142/jvs.2013.14.1.69

Abstract

The use of mesenchymal stem cells (MSCs) has emerged as a potential new treatment for myocardial infarction. However, the poor viability of MSCs after transplantation critically limits the efficacy of this new strategy. The expression of microRNA-210 (miR-210) is induced by hypoxia and is important for cell survival under hypoxic conditions. Hypoxia increases the levels of hypoxia inducible factor-1 (HIF-1) protein and miR-210 in human MSCs (hMSCs). miR-210 positively regulates HIF-1alpha activity. Furthermore, miR-210 expression is also induced by hypoxia through the regulation of HIF-1alpha. To investigate the effect of miR-210 on hMSC survival under hypoxic conditions, survival rates along with signaling related to cell survival were evaluated in hMSCs over-expressing miR-210 or ones that lacked HIF-1alpha expression. Elevated miR-210 expression increased survival rates along with Akt and ERK activity in hMSCs with hypoxia. These data demonstrated that a positive feedback loop involving miR-210 and HIF-1alpha was important for MSC survival under hypoxic conditions.

Keyword

cell survival; human mesenchymal stem cells; hypoxia; hypoxia inducible factor-1; microRNA-210

MeSH Terms

Cell Survival
Cobalt
Gene Expression Regulation/*physiology
Humans
Hypoxia-Inducible Factor 1, alpha Subunit/genetics/*metabolism
Mesenchymal Stromal Cells/drug effects/metabolism/*physiology
MicroRNAs/*metabolism
Oxygen/pharmacology
*Oxygen Consumption
RNA, Small Interfering/metabolism
Hypoxia-Inducible Factor 1, alpha Subunit
MicroRNAs
RNA, Small Interfering
Cobalt
Oxygen

Figure

• 

  Fig. 1 Expression of hypoxia inducible factor (HIF)-1α in human mesenchymal stem cells (hMSCs) under hypoxic conditions. hMSCs were treated with 250 µM CoCl2 for 24 h and incubated in a hypoxic chamber for 24 h. (A) Expression of HIF-1α was detected by immunoblotting. (B) The ratio of HIF-1α to β-actin band density values. Results are expressed as the mean ± SE for three independent experiments performed in triplicate. *p < 0.01 and **p < 0.05 vs. the control.

• 

  Fig. 2 Expression of microRNA-210 (miR-210) with hypoxia or CoCl2 treatment. miR-210 expression was measured by real-time PCR. Expression of endogenous miR-210 was detected under hypoxic conditions over time or in the presence with 250 µM CoCl2 for 24 h. Results are expressed as the mean ± SE for three independent experiments performed in triplicate. *p < 0.01 and **p < 0.05 vs. the control.

• 

  Fig. 3 Effect of miR-210 over-expression on HIF-1α protein stability in hMSCs. (A) Transfection efficiency of the miR-210 mimic was monitored by real-time PCR. (B) Expression of HIF-1α was detected by immunoblotting. Cells transfected with the miR-210 mimic were incubated under normoxic or hypoxic conditions. Results are expressed as the mean ± SE for three independent experiments performed in triplicate. *p < 0.01 and **p < 0.05 vs. the control.

• 

  Fig. 4 Effect of HIF-1α down-regulation on the induction of miR-210 expression under hypoxic conditions. hMSCs were transfected with HIF-1α-specific small interfering RNA (si-HIF-1α) at a final concentration of 100 nM and incubated in a hypoxia chamber for 12 h. (A) Expression of HIF-1α and β-actin was detected by immunoblotting. (B) miR-210 expression was measured by real-time PCR. Results are expressed as the mean ± SE for three independent experiments performed in triplicate. *p < 0.01 vs. the control.

• 

  Fig. 5 Effect of miR-210 and HIF-1α on the survival of hypoxic hMSCs. After transfection with the miR-210 mimic, the survival of hMSCs was measured with a CCK-8 assay. Data presented a typical experiment conducted more than three times. Results are expressed as the mean ± SE. *p < 0.01 and **p < 0.05 vs. the control.

• 

  Fig. 6 Effect of miR-210 and HIF-1α on cell survival-related signaling in hypoxic hMSCs. Anti-apoptotic signaling was detected by immunoblotting. Cells were incubated under hypoxic conditions for 24 h. Results are expressed as the mean ± SE for three independent experiments performed in triplicate. *p < 0.01 and **p < 0.05 vs. the control.

• 

  Fig. 7 Relationship between HIF-1α and miR-210 under hypoxic conditions. The relation of HIF-1α and miR-210 results in a positive feedback regulatory loop. As shown in this figure, miR-210 acts to induce and maintain the expression of HIF-1α protein. CASP8AP2: caspase 8-associated protein 2.

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