Circular RNAs correlate with DNA damage in ageing human placental tissue and in stillbirth

Abstract

AbstractUnexplained stillbirth is hypothesised to occur due to premature placental ageing, with unexpected deterioration of placental function for gestational age. Circular RNAs (circRNAs) are enzyme resistant RNA molecules that accumulate in ageing tissues. Furthermore, circRNAs bind gDNA directly, forming circRNA:DNA complexes which can induce DNA breaks. This study investigated circRNA accumulation with gestational age in healthy and stillbirth placentae and determined whether circRNAs directly interact with placental DNA causing DNA damage.Placenta samples (n=60 term uncomplicated; n=4 unexplained stillbirth, 23, 26, 31, 34 weeks’ gestation) were assessed for DNA damage using an alkaline Comet Assay. Expression levels of 6 candidate circRNAs (circ_0009000, circ_0024157, circ_0061017, circ_0036877, circ_0054624 and circ_0111277), and their linear transcripts, were quantified using qPCR. Physical interaction of candidate circRNAs with DNA was confirmed by DNA:RNA ImmunoPrecipitation (DRIP). The effect of circ_0009000 knockdown in HEK293T cells was assessed following transfection with either a siRNA (designed to knockdown circ_0009000) or a scrambled siRNA control, at 5, 10 and 20 nM final concentrations using Lipofectamine RNAiMax. DNA damage was assessed by Comet Assay. Compared with earlier gestations (37, 38, 39 and 40 weeks’), placental DNA damage and expression of all 6 candidate circRNAs, but not their linear transcripts, were increased in 40 and 41+ weeks’ gestation samples, and in stillbirth.DRIP-qPCR signal size was significantly larger in term placentae than in enzyme-treated controls, confirming that all candidate circRNA loci bind to placental DNA.Depletion of circ_0009000 by specific siRNA in HEK293T cells, significantly reduced DNA damage compared to control.Stillbirth placentae show accelerated ageing with premature accumulation of candidate circRNAs (first evidence in humans) at levels consistent with older gestation tissue. Importantly, these circRNAs bind to DNA and circ_0009000 causes DNA breaks in placenta. Therefore, circRNAs (circ_0009000, circ_0024157, circ_0061017, circ_0036877, circ_0054624 and circ_0111277) play a role in placental ageing and associate with stillbirth, likely via DNA damage.Graphical Abstract