Hypoxia-induced down-regulation of microRNA-449a/b impairs control over targeted SERPINE1 (PAI-1) mRNA - a mechanism involved in SERPINE1 (PAI-1) overexpression

After publication of our article [1], we realized the need for posting a correction note in order to prevent i) overinterpretation of some results by the readers and ii) concerns about potentially unintended misguidance by the authors.

Array-based gene expression analysis (mRNA, miRNA, respectively) was performed in 2 independent experiments resulting in 2 values for differentially expressed targets (Figures one and three). The resulting error bars therefore appear symmetrical.
In the published figures two, four, five and six, data from 1 experiment, i.e. 2 measurements, are shown, likewise resulting in symmetrical error bars. These figures are based on data sets underlying version 1 of the submitted manuscript. Version 2 of the manuscript for publication should contain figures two, four, five, and six (figures 1,2,3, and 4 in this correction) and the underlying data sets from 3 independent experiments with 2 measurements (values) per experiment. Unfortunately, internal errors eventually have led to discrepancies between intended illustration of data sets and the published ones.
We have now incorporated the valid figures and figure legends. The underlying data set is shown in the supplement of this correction note (additional file 1). The corrected figures five and six (figures 3 and 4 here) now need particular attention because a subtle interpretation is necessary.
When we transfected either miRNA-449a/b inhibitors or mimics, we noted the expected decrease or increase of the miRNA-449 subtype under investigation ( When hypoxic fibroblasts were transfected with miRNA-449a/b inhibitors, SERPINE1 (PAI-1) mRNA was additionally increased up to 3-fold while miRNA-449a/b mimics showed no effect with the concentrations used. The "stumbling block" in the original figure six was our aim to illustrate the transfection-independent effect of hypoxia-induced SERPINE1 (PAI-1) mRNA Unfortunately, it could also suggest a false-positive effect of miRNA-449a/b mimics, i.e. notable down-regulation of SERPINE1 (PAI-1) mRNA. It must be emphasized that the latter effect is not demonstrable because the negative control showed almost identical SERPINE1 (PAI-1) mRNA levels. We therefore revised Figure six (figure 4 here) which now shows the apparent effect of miRNA-449a/b inhibitors but not of the mimics. We meanwhile resigned from insisting on a statistically robust effect of miRNA-449a/b inhibitors on PAI-1 mRNA expression. Dependent on the statistical test used we were able to demonstrate a tendency only for the miRNA-449b inhibitor at 50 nM (p < 0.05). The biological significance of the hypoxia-dependent effect on miRNA-449a/b levels demonstrable in vitro remains to be further investigated in vivo. As shown for organ fibrosis by using kidney allograft remodelling (Figure eight), SERPINE1 (PAI-1) mRNA was increased whilst miRNA-449a/b species were decreased. We do not know the extent of the miRNA-449a/b contribution to SERPINE1 (PAI-1) mRNA level in hypoxic environments in remodelled tissues. However, we believe that, based on the in vitro data shown, a subtle mechanism contributes to higher SERPINE1 (PAI-1) mRNA levels through down-regulated miRNA-449a/b. Until we were given the opportunity to prepare this correction note, we reproduced the hypoxic cell culture experiments in a standardized fashion. Our lab was recently equipped with a Hypoxystation H35 from Don-Whitley and instead of using the "Anaeropack for cell culture" (which, by definition, produces an almost anoxic condition without monitoring the definite concentration), we now culture cells under controlled and monitored oxygen concentrations.

Transfection of miRNA Inhibitor/ miRNA
Because of the continuously adjustable oxygen concentration in the hypoxystation, we found that 5 percent oxygen or less is needed to induce a decrease of miRNA-449a/b expression levels in different cell lines. Interestingly, oxygen concentrations of 1 percent decreased the miRNA-449a/b level more than 5 percent did. In contrast, SERPINE1 (PAI-1) mRNA apparently needs oxygen levels below 1 percent to show a remarkable upregulation. Analyses were not restricted to fibroblasts but also included leukemic and solid tumor cell lines. Since this correction note is not a platform for presentation of new data, we only wish to underline our still ongoing activities in this field.
We offer our sincere apologies to the scientific community for any confusion or misleading interpretation we may have caused.