miRNA-target gene-signaling pathway regulatory network and PCR validation. (A) miRNA-target gene-signaling pathway regulatory network. Diamonds and nodes represent miRNAs and target genes, among which the orange diamond and blue nodes represent an miRNA-mRNA pair validated by a dual luciferase experiment. Red polygons represent signaling pathways. Grey lines represent potential regulatory associations between miRNAs and their target genes. (B) Venn diagram of miRNAs differentially expressed at two time-points and included in the miRNA-target gene-signaling pathway regulatory network. Overlap between the two circles represent five miRNAs included in the network that are differentially expressed at two time-points and validated by PCR. (C) Relative expression levels of selected miRNAs in N and A groups. Relative miRNA expression is presented as the mean ± standard deviation. *P<0.05; **P<0.01. miR/miRNA, microRNA; PCR, polymerase chain reaction; N, normal; A, anorectal malformations; GD, gestational day.