miR-200b downregulates Kruppel Like Factor 2 (KLF2) during acute hypoxia in human endothelial cells

Abstract

The role of microRNAs in controlling angiogenesis is recognized as a promising therapeutic target in both cancer and cardiovascular disorders. However, understanding a miRNA's pleiotropic effects on angiogenesis is a limiting factor for these types of therapeutic approaches. Using genome-wide next-generation sequencing, we examined the role of an antiangiogenic miRNA, miR-200b, in primary human endothelial cells. The results indicate that miR-200b has complex effects on hypoxia-induced angiogenesis in human endothelia and importantly, that many of the reported miR-200b effects using miRNA overexpression may not be representative of the physiological role of this miRNA. We also identified the antiangiogenic KLF2 gene as a novel target of miR-200b. Our studies indicate that the physiological changes in miR-200b levels during acute hypoxia may actually have a proangiogenic effect through Klf2 downregulation and subsequent stabilization of HIF-1 signaling. Moreover, we provide a viable approach for differentiating direct from indirect miRNA effects in order to untangle the complexity of individual miRNA networks.

Keywords: HIF-1; HIF-2; HUVEC; KLF2; Micro-RNA 200b; hsa-miR-200b-3p.

Figure 1

Regulation of miR-200b during hypoxia in primary HUVECs. (A) The miR-200b levels were monitored in qRT-PCR experiments. The results from 3 independent experiments (n=12) are plotted normalized to RNU44 and RNU48 RNA levels and expressed as a fold-change over the normoxic control. (B) HUVECs were transfected with miR-200b mimic or antagomiR, and the miRNA levels were monitored in normoxic conditions and after 4 h of exposure to hypoxia. miRNA levels from 2 independent experiments (n=8) are plotted normalized to RNU44 and RNU48 RNA levels and expressed as a fold change over normoxic control. Error bars represent standard deviations. Significant changes (p<0.05) are marked with an asterisk.

Figure 2

Acute hypoxia induces dynamic changes in the protein expression profile of the Klf2 in HUVECs. (A) Hypoxia induces dynamic changes of protein levels of Klf2, Hif-1α, and Hif-2α. The protein levels of were detected with SDS-PAGE and Western Blot and related to total protein levels. 2 individual samples (4 µg of total protein per lane) were tested for each time point and the experiments were repeated twice. (B) The KLF2 mRNA levels were monitored in qRT-PCR experiments. The results from 3 independent experiments (n=12) are plotted normalized to 18S rRNA levels and expressed as a fold-change over the normoxic control. Error bars represent standard deviations. Significant changes (p<0.05) are marked with an asterisk.

Figure 3

miR-200b alters the expression of KLF2. (A) HUVECs were transfected with miR-200b mimic or antagomiR, and the mRNA levels were monitored in normoxic conditions and after 4 h exposure to hypoxia. KLF2 mRNA levels from 3 independent experiments (n=12) are plotted normalized to 18S rRNA or TBP mRNA levels and expressed as a fold change over the normoxic control. Significant changes (p<0.05) are marked with an asterisk. (B) The corresponding changes of Klf2 protein levels were detected with SDS-PAGE and Western Blot and normalized to the β-Actin and 2 individual samples (4 µg of total protein per lane) were tested for each treatment and the experiments were repeated twice.

Figure 4

miR-200b binds to predicted target sequence in the KLF2 3’UTR (A).The RNAhybrid predicted miR-200b target site at 3' UTR of KLF2 mRNA is presented at the top right. HUVECs were transfected with KLF2 target sequence-specific target protector and/or the miR-200b analog (Mimic 200b) and exposed to hypoxia for 4 h. The KLF2 mRNA levels were monitored in qRT-PCR experiments. The KLF2 levels results from 2 independent experiments (n=8) are plotted normalized to 18S rRNA levels and expressed as a fold change over the transfection control. Significant changes (p<0.05) are marked with an asterisk. (B). The corresponding changes of Klf2 protein levels were detected with SDSPAGE and Western Blot and normalized to the β-actin levels 2 µg of total protein per lane was loaded for each sample and the experiments were repeated twice.