The role of oxygen in regulating microRNAs that control the placental renin-angiotensin system

The human placental renin-angiotensin system (RAS) genes encoding for prorenin (REN), angiotensinogen (AGT), prorenin receptor (ATP6AP2) and angiotensin II type I (AGTR1) receptor are upregulated in early gestation and affect placental development. At this time, the placental oxygen tension is at its lowest (1-3%). Some miRNAs predicted to target the RAS are downregulated in early gestation. Our hypothesis is that the low oxygen tension in early pregnancy suppresses expression of miRNAs that target the placental RAS; thus expression of these RAS genes are upregulated. Ten miRNAs predicted to target RAS mRNAs were downregulated in response to 1% vs. 20% oxygen, and six miRNAs were decreased in cells cultured in 1% vs. 5% oxygen. HTR-8/SVneo cells, a human extravillous trophoblast cell line, were cultured in 1%, 5% and 20% oxygen tensions (to reflect the oxygen tensions of the chorionic plate, decidua and standard culture conditions respectively) for 48 h (n=3 in triplicate). Any differences in expression of miRNAs between these groups were determined. Affymetrix miRNA microarray analysis was conducted, which detected up to 2006 miRNAs. Statistically different levels of expression were determined. MiRNAs predicted to target the placental RAS were identified using a variety of databases (mirbase.org, mirdb.org, targetscan.org, and targetexplorer.ingenuity.com). Differences in miRNA expression measured on the microarray were confirmed for some miRNAs by RT-PCR (qPCR, n=3 in triplicate for all groups).