Rno_circ_0005139 regulates apoptosis by targeting Wnt5a in rat anorectal malformations

ARTICLE HIGHLIGHTS

Research background

Anorectal malformations (ARM) are common in pediatric surgery. During normal embryonic development of the anus and rectum, the Wnt signaling pathway is one of the most mature and important pathways in ARM, but its upstream regulatory mechanism is not clear. Due to its strong stability and high conservation, circular RNA (circRNA) is considered to be the most competitive microRNA (miRNA) "sponge", which competitively binds to the 3'-non-coding region of the common target gene and regulates the expression process of the target gene. circRNA is rarely reported in the research of ARM, and circRNA acts on miRNA, and then regulates the expression of Wnt signaling pathway in ARM, and affects cell proliferation and apoptosis, which are of research significance.

Research motivation

ARM is often combined with multiple system malformations. Although they can be treated surgically, children with multiple system malformations often require long-term multidisciplinary treatment, and some children even need psychological treatment. The quality of life is poor, and the prognosis is not ideal. The mechanism of ARM is still unclear. Therefore, an in-depth study of circRNA and the upstream of the Wnt genes in ARM will provide a new research direction for ARM and a new approach to prenatal intervention therapy to improve the children’ quality of life.

Research objectives

To investigate differentially expressed circRNAs and mRNAs in a rat ARM model and identify the mechanism of rno_circ_0005139 targeting Wnt5a in proliferation and apoptosis.

Research methods

We chose 66 pregnant Wistar rats and randomly divided them into two groups: Ethylenethiourea-induced ARM group and control group. A total of 652 embryos was harvested by cesarean delivery and anorectal tissue was taken on embryonic day 16 (E16), 17 (E17), 19 (E19) and 21 (E21). RNA sequencing and gene microarray analysis were used to identify differentially expressed circRNAs and mRNAs in 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. 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 performed to verify their functional consistency.

Research results

We found 38 upregulated and 42 downregulated circRNAs on E17, and 301 mRNAs were upregulated and 256 downregulated in the ARM on E17. We also observed 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. A luciferase experiment showed that rno_circ_0005139 was a sponge for miR-324-3p, which negatively regulated Wnt5a expression. MiR-324-3p expression was significantly higher in ARM group anorectal tissues than in normal control tissues on E17 and E19; Wnt5a expression showed the opposite trend. The knockdown of rno_circ_0005139 promoted cell apoptosis. In addition, a miR-324-3p inhibitor attenuated the effects of rno_circ_0005139 knockdown on ARM development and cell apoptosis.

Research conclusions

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 are potential therapeutic targets for ARM.

Research perspectives

There are a large number of circRNAs in ARM, and in terms of the mechanism of rno_circ_0005139/miR-324-3p/Wnt5a, the specific regulatory mechanism of circRNAs is the focus of future research. In line with this, future directions should include studies exploring the diagnostic and therapeutic potential of circRNAs.