Oral Presentation ESA-SRB-ANZBMS 2024 in conjunction with ENSA

The clock is ticking: circular RNAs, placental ageing and stillbirth (#222)

Anya L Arthurs 1 2 , Matilda Jackson 3 , Dylan McCullough 1 2 , Hamish S Scott 3 , Christopher P Barnett 4 , Stuart Webb 1 3 , Melanie D Smith 1 2 , Tanja Jankovic-Karasoulos 1 2 , Gustaaf A Dekker 5 , Claire T Roberts 1 2
  1. Flinders University, Bedford Park, SA, Australia
  2. Flinders Health and Medical Research Institute, Adelaide, SA, Australia
  3. SA Pathology, Adelaide, SA, Australia
  4. Women's and Children's Hospital, North Adelaide, SA, Australia
  5. University of Adelaide, Adelaide, SA, Australia

Unexplained stillbirth is hypothesised to occur due to premature placental ageing, with unexpected deterioration of placental function for gestational age(1). Circular RNAs (circRNAs) are enzyme resistant RNA molecules that accumulate in ageing tissues(2-4). Furthermore, circRNAs bind gDNA directly, forming circRNA:DNA complexes which induce DNA breaks(5). Given the above, we 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 (identity commercial in confidence), 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_A knockdown in HEK293T cells was assessed following transfection with siRNA (designed to knockdown circ_A) or a scrambled siRNA control, at 5, 10 and 20 nM final concentrations using Lipofectamine RNAiMax. DNA damage was assessed by Comet Assay. Appropriate statistical analyses were undertaken (SPSS).

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_A by 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_A causes DNA breaks in placenta. Therefore, circ_A plays a role in placental ageing and associates with stillbirth, likely via DNA damage.

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