P0738 - Hypoxia induction of anti-inflammatory placenta-specific miRNAs

Increased levels of placental pro-inflammatory mediators have been implicated in the pathogenesis of preterm labor. Our prior work showed placental hypoxia induces pro-inflammatory mediators known to trigger preterm labor unless countered by placental production of anti-inflammatory molecules. Placental miRNAs play an essential role in maintaining immunologic homeostasis in the intrauterine environment, which is necessary for progression of normal term pregnancies. In particular, the placenta-specific miR-519c is a key anti-inflammatory miRNA that maintains placental immune tolerance. However, the effect of hypoxia on placental miRNA expression has not been investigated. The objective of this study was to determine if human placental explants cultured at low oxygen tensions will have altered expression of placental miRNAs. Furthermore, the role of miR-519c in reversing placental inflammation was investigated.

Placental explant cultures from second-trimester elective abortions and term pregnancies were incubated at oxygen concentrations of either: 5% (physiologic placental PO2), 1% (hypoxic condition) or 21% (hyperoxic condition) for 24 hours. Placental tissue (n=6 for 2nd trimester, n=4 for term) and culture medium (n=5 for 2nd trimester, n=4 for term) were used to obtain RNA, which was reverse-transcribed using the miScript Reverse II RT Kit and assayed by miScript SYBR Green PCR. Relative miRNA expression levels were expressed as dCT values using RNU-6 as an internal control. Transfection of human monocyte cell lines with miR-519c mimic, followed by incubation at the oxygen concentrations described above, was conducted to observe the influence miR-519c on TNF-a production under hypoxic conditions. The nonparametric Friedman test was used to analyze the difference between miRNA expression levels in tissue and culture media from placental explant experiments. A p-value <0.05 was required for significance.

We observed a statistically significant difference in miR-519c expression across different oxygen concentrations for term tissue (p=0.0046) and media (p=0.0046), and second-trimester tissue (p=0.0289) and media (=0.0394). Interestingly, the expression of miR146a, an anti-inflammatory miRNA not specific to the placenta, was not different to a statistically significant degree between tissue and media for both term and second trimester samples. Additionally, preliminary data suggests transfection with miR-519c mimic may inhibit TNF-a production under hypoxic conditions in monocyte cell lines.

These results demonstrate that hypoxic conditions significantly increased placental miR-519c expression in 2nd trimester and term placentas and culture media; on the other hand, miR-519c expression trended down with increased oxygen tension. Furthermore, human monocyte cells transfected with miR-519c mimic may have inhibited TNF-a production, however, further experiments are needed to validate this claim. To our knowledge, these results are the first to demonstrate the role of hypoxia in regulating miR-519c, a key anti-inflammatory, placenta-specific miRNA. We speculate that the placenta will have limited capacity to induce miR-519c, in response to hypoxic or other inflammatory insults, in inflammation-mediated pathologic pregnancies.