Abstract
Hypoxia enhances the proliferation and migration of adipose-derived stem cells (ASCs) via the generation of reactive oxygen species (ROS). Therefore, this study primarily investigated whether or not ROS generation could regulate microRNA-210 (miR-210) expression, and increase proliferation/migration of ASCs. In addition, we tried to identify the signaling pathways involved in miR-210 upregulation and the direct target genes of miR-210 that mediate these functions. Various sources of ROS generation such as hypoxia, antimycin, rotenone, and platelet-derived growth factor (PDGF)-BB upregulated miR-210 expression, and increased the proliferation/migration of ASCs. There is a positive feed-forward loop between ROS generation and miR-210, and miR-210 itself increases ROS generation by downregulation of iron–sulfur cluster scaffold homolog 2 (ISCU2). Although hypoxia-inducible factor-1α was not involved in miR-210 expression, pharmacological or small interfering RNA (siRNA)-driven inhibition of Akt and ERK1/2 molecules reduced miR-210 expression. Transfection of siRNAs of NF-κB and Elk1 also reduced miR-210 expression, indicating that these signaling pathways mediate miR-210 upregulation. Protein tyrosine phosphatase, non-receptor type 2 (PTPN2) was selected for miR-210 target gene, and it was downregulated by ROS generators or miR-210 mimic treatment. PTPN2 was first proven to be a direct miR-210 target in luciferase activity assay, and pharmacological inhibition or overexpression of PTPN2 regulated the proliferation and migration of ASC. In conclusion, ROS generation from diverse sources induces miR-210 expression in ASCs via PDGFR-β, Akt and ERK pathways. Transcription of miR-210 expression is regulated by NF-κB and Elk1, and miR-210 increases the proliferation and migration of ASCs via ISCU2 and PTPN2 downregulation.
Highlights
Hypoxia influences cellular functions, including metabolism, survival, proliferation, migration and angiogenesis
We confirmed the miR-210 expression using quantitative real-time PCR (Q-PCR), and found that hypoxia (2%) significantly increased the miR-210 expression in a time-dependent manner (Figure 1a; Po0.01). miR-210 expression was not altered until 6 h under hypoxia
Hypoxia-induced expression of miR-210 was attenuated by the reactive oxygen species (ROS) scavenger, such as N-acetyl-cysteine (NAC), indicating that hypoxia-increased miR-210 expression is mainly mediated by ROS generation (Figure 1b; Po0.01)
Summary
Hypoxia influences cellular functions, including metabolism, survival, proliferation, migration and angiogenesis. All of these cellular functions are directly and indirectly controlled by a master transcription factor called hypoxia-inducible factor-1a (HIF-1a).[9,10] In response to hypoxia, HIF-1a levels increase a specific set of microRNA molecules called miR-210. This unique microRNA is highly expressed in hypoxic cells and tissues, and is reportedly regulated by HIF-1a stabilization.[11,12,13]. There are some evidences that expression of miR-210 is independent of HIF-1a pathway, but it was regulated by Akt- or TNF-a-related pathways.[19,20] this study investigated: (1) Whether or not ROS generation regulates miR-210 expression, and increase proliferation/migration of ASCs? (2) Which signaling pathway(s) and transcription factor(s) are involved in the upregulation of miR-210? (3) What is (are) miR-210 target gene(s) involved in proliferation and migration of ASCs? We first found that ROS generation induces miR-210 expression, and identified the novel transcription factors and target genes of miR-210 in this study
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