Circular RNAs accumulate in ageing human placental tissue and in stillbirth, leading to DNA damage and cellular senescence

Abstract

Structured Abstract: Background: Unexplained stillbirth may 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 and genomic instability. Objectives: This study investigated tissue ageing and circRNA accumulation with gestational age in healthy and stillbirth placentae, and determined whether circRNAs directly interact with placental DNA causing DNA damage and cellular senescence. Study design: Placenta samples (n=60 term uncomplicated; n=4 unexplained stillbirth, 23, 26, 31, 34 weeks’ gestation) were assessed. Abundance of 7 candidate circRNAs (circ_0009000, circ_0024157, circ_0061017, circ_0036877, circ_0054624, circ_0111277 and circ_0000284), and their linear transcripts, was quantified using qPCR. Total RNA, in the presence and absence of RNase R and RNase H1, was determined using the Qubit fluorimeter. Physical interaction of candidate circRNAs with DNA was confirmed by DNA:RNA ImmunoPrecipitation (DRIP)-qPCR. Telomere length was assessed using real-time PCR. Relative abundance of senescence-associated genes was quantified using qPCR. DNA damage was assessed using an alkaline Comet Assay. Patient-derived trophoblast stem cells (TSCs) differentiation into syncytiotrophoblasts or extravillous trophoblasts was confirmed using immunofluorescence microscopy. The effect of circ_0000284 knockdown was assessed following transfection with either a siRNA (designed to knockdown circ_0000284) or a scrambled siRNA control, at 5, 10 and 20 nM final concentrations using Lipofectamine RNAiMax. Abundance of circRNAs in maternal blood sampled between 15-16 weeks’ gestation (n=12 control, n=6 women who went on to have a stillbirth) was determined using qPCR. Appropriate statistical analyses were undertaken (SPSS). Results: Placental DNA damage, senescence and expression of 7 candidate circRNAs, but not their linear transcripts, were increased in 40 and 41+ weeks’ gestation samples, and in stillbirth, compared with earlier gestations (37-39 weeks’). 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. Abundance of circRNA was significantly decreased with the addition of RNase H1, compared with all healthy gestation samples, indicating that stillbirth placentae may lack RNase H1. Telomere length is shorter in placentae from stillbirths compared with healthy 37 weeks’ placentae. Depletion of circ_0000284 by specific siRNA in primary cells significantly reduced DNA damage and increased expression of senescence-associated genes compared to control. Abundance of candidate circRNAs are increased in maternal blood at 16 weeks’ gestation for women who went on to have a stillbirth compared with women who had live births. Conclusions: Stillbirth placentae show accelerated ageing with shortened telomeres, premature DNA breaks, increased cellular senescence and accumulation of candidate circRNAs, at levels consistent with older gestation tissue. These circRNAs bind to DNA in the placenta, and circ_0000284 knockdown reduces DNA breaks and senescence in primary placental cells. Therefore, circRNAs play a role in placental ageing and associate with stillbirth, likely via decreased RNase H1 abundance, preventing circRNA degradation and facilitating circRNA accumulation, and subsequent circR-loop formation. circRNAs present a viable method of stillbirth risk screening. Graphical Abstract: AJOG at a Glance: This study was conducted to confirm premature placental ageing in cases of unexplained stillbirth, and to investigate the role of circular RNAs in the process of placental ageing. Circular RNAs accumulate in ageing healthy placenta and accumulate prematurely in stillbirth placentae. Circular RNAs bind directly to placental DNA, inducing DNA breaks and cellular senescence, thereby contributing to overall functional decline of the placenta, reducing its ability to support the fetus. Circular RNAs in maternal blood represents a novel screening tool for detection of stillbirth risk. Premature placental ageing has previously been associated with stillbirth, however circular RNAs have never before been implicated in this process. Circular RNA accumulation in ageing tissues has been shown in model species (i.e. C. elegans, Drosophila Melanogaster , mice) but this is the first evidence in human tissues and in the placenta.