Microarray analysis of miRNAs during hindgut development in rat embryos with ethylenethiourea-induced anorectal malformations

Anorectal malformations (ARMs) are one of the most common congenital malformations of the digestive tract; however, the pathogenesis of this disease remains to be fully elucidated. MicroRNAs (miRNAs) are important in gastrointestinal development and may be involved in the pathogenesis of ARMs. The present study aimed to profile miRNAs and examine their potential functions in rats with ethylenethiourea (ETU)-induced ARMs. Pregnant Wistar rats (n=36) were divided randomly into ETU-treated and control groups. The rats in the ETU-treated group were gavage-fed 1% ETU (125 mg/kg) on gestational day 10 (GD10), whereas the control group rats received a corresponding dose of saline. Embryos were harvested by cesarean section on GD14, GD15 and GD16. Hindgut tissue was isolated from the fetuses for RNA extraction and microarray analysis, followed by bioinformatics analysis and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) validation. Overall, 38 miRNAs were differentially expressed (all upregulated) on GD14, 49 (32 upregulated and 17 downregulated) on GD15, and 42 (all upregulated) on GD16 in the ARM group compared with the normal group. The top 18 miRNAs with |log(2)(fold change)| >4.25 were selected for further bioinformatics analysis. Among these miRNAs, five were differentially expressed at two time-points and were involved in ARM-associated signaling pathways. The RT-qPCR analysis revealed that three miRNA (miR), miR-125b-2-3p, miR-92a-2-5p and miR-99a-5p, were significantly differentially expressed in rats with ARMs compared with the normal group. In conclusion, the results suggested that the differential expression of miR-125b-2-3p, miR-92a-2-5p and miR-99a-5p during key time-points of anorectal formation in rats may have functions in the pathogenesis of ARM.