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

Abstract

Background: Unexplained stillbirth may occur owing to premature placental aging, with an unexpected deterioration of placental function for the gestational age. Circular RNAs are enzyme-resistant RNA molecules that accumulate in aging tissues. Furthermore, circular RNAs bind to genomic DNA directly, forming complexes that can induce DNA breaks and genomic instability.

Objective: This study investigated tissue aging and circular RNA accumulation with gestational age in healthy and stillbirth placentae and determined whether circular RNAs directly interact with placental DNA, causing DNA damage and cellular senescence.

Study design: Placenta samples (n=60 term uncomplicated; n=4 unexplained stillbirth, at 23, 26, 31, and 34 weeks' gestation) were assessed. The abundance of 7 candidate circular RNAs and their linear transcripts was quantified. The physical interaction of candidate circular RNAs with DNA was confirmed. Telomere length, relative abundance of senescence-associated genes, and DNA damage were assessed. Patient-derived trophoblast stem cell differentiation into syncytiotrophoblasts or extravillous trophoblasts was confirmed before circ_0000284 knockdown. The abundance of circular RNAs in maternal blood sampled between 15 and 16 weeks' gestation (n=12 control, n=6 women who went on to have a stillbirth) was determined using quantitative polymerase chain reaction. Appropriate statistical analyses were undertaken (SPSS).

Results: Placental DNA damage, senescence, and expression of 7 candidate circular RNAs, but not their linear transcripts, were increased in 40 and 41+ weeks' gestation samples and in stillbirth compared with earlier gestations (37-39 weeks). DNA:RNA immunoprecipitation-quantitative polymerase chain reaction signal size confirmed that all candidate circular RNA loci bind to placental DNA. The abundance of circular RNA 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 than in healthy 37 weeks' gestation placentae. Depletion of circ_0000284 by specific small interfering RNA in primary cells significantly reduced DNA damage and increased expression of senescence-associated genes compared with the control. The abundance of candidate circular RNAs is increased in maternal blood at 16 weeks' gestation for women who went on to have a stillbirth compared with women who had live births.

Conclusion: Stillbirth placentae show accelerated aging with shortened telomeres, premature DNA breaks, increased cellular senescence, and accumulation of candidate circular RNAs at levels consistent with older gestation tissue. These circular RNAs bind to DNA in the placenta, and circ_0000284 knockdown reduces DNA breaks and senescence in primary placental cells. Therefore, circular RNAs play a role in placental aging and are associated with stillbirth, likely via decreased RNase H1 abundance, preventing circular RNA degradation and facilitating circular RNA accumulation and subsequent circular RNA:DNA complex formation. Circular RNAs may present a viable method of stillbirth risk screening.

Keywords: DNA; aging; circular RNA; double-stranded breaks; placenta; pregnancy; senescence; stillbirth; telomere.