Rno_circ_0005139 regulates apoptosis by targeting Wnt5a in rat anorectal malformations

The molecular mechanisms underlying anorectal malformations (ARM) are not fully established. Circular RNAs (circRNAs) are new born non-coding RNAs, and their role in ARM is unclear. We assumed that rno_circ_0005139 influences apoptosis and proliferation by acting as a miR-324-3p sponge, and downregulating Wnt5a in ARM.

To identify the differential expression of circRNAs and mRNAs in a rat ARM model.

Sixty-six pregnant Wistar rats were randomly divided into two groups: ARM group (2-imidazolidinethione-induced) and control groups. Embryos were harvested by cesarean delivery, and anorectal tissue was taken on embryonic days 16 (E16), 17 (E17), 19 (E19), and 21 (E21). RNA sequencing and gene microarray analysis was used to identify differentially expressed circRNAs and mRNAs in the ARM in a rat model. We selected 6 circRNAs and 3 mRNAs in the Wnt signal pathway from the result of the RNA sequencing and gene microarray analysis, and quantitative reverse transcription polymerase chain reaction was performed to evaluate their tissue expression. According to bioinformatics prediction, rno_circ_0005139 acted as a miR-324-3p sponge to regulate the expression of Wnt5a. We chose rno_circ_0005139 and Wnt5a as the final candidates. We tested the function of rno_circ_0005139 and the binding sites between rno_circ_0005139 and miR-324-3p, miR-324-3p and Wnt5a by luciferase assays. Co-transfection of rno_circ_0005139 and miR-324-3p was to verify their functional consistency.

We identified 38 upregulated and 42 downregulated circRNAs on E17 (P < 0.05), and 301 mRNAs were upregulated and 256 downregulated in the ARM on E17 (P < 0.05, fold-change > 2.0). We found that rno_circ_0006880 and rno_circ_0011386 were upregulated, whereas rno_circ_0000436, rno_circ_0005139, rno_circ_0009285, rno_circ_0014367, Wnt5a, Wnt10b, and Wnt2b were downregulated in ARM tissues. According to bioinformatics prediction, rno_circ_0005139 acted as a miR-324-3p sponge to regulate the expression of Wnt5a. We chose rno_circ_0005139 and Wnt5a as the final candidates. Because the role and molecular mechanism of rno_circ_0005139 are poorly understood, its effect on apoptosis and proliferation was investigated by in vitro plasmid transfection. A luciferase experiment showed that rno_circ_0005139 could bind with miR-324-3p, which negatively regulated Wnt5a expression. The expression of miR-324-3p was significantly higher in ARM anorectal tissues than that in control group on E17 and E19; Wnt5a expression showed the opposite trend. In addition, a miR-324-3p inhibitor attenuated the effects of rno_circ_0005139 knockdown on ARM development.

Rno_circ_0005139 influences cell proliferation and apoptosis by acting as a miR-324-3p sponge, thereby downregulating Wnt5a in ARM. Accordingly, rno_circ_0005139, miR-324-3p, and Wnt5a could be targeted therapeutic factors for ARM.