Upregulation of PPPDE1 contributes to anorectal malformations via the mitochondrial apoptosis pathway during hindgut development in rats.

Congenital anorectal malformations (ARMs) are among the most prominent deformities of the gastrointestinal tract; however, their precise aetiology remains obscure. Immunohistochemistry demonstrated that, in the ARM group, the PPPDE1-positive cells were widely distributed in the hindgut epithelial tissue from GD13 to GD16. Immunofluorescence revealed that most TUNEL-, Bax-, and Cytochrome C (Cyt C)-positive cells overlapped with PPPDE1-positive cells in the urorectal septum (URS). Western blotting and quantitative real-time RT-PCR revealed that PPPDE1 levels were significantly higher in the ARM group from GD13 to GD14 (p < 0.05). IEC-6 cells were transfected with PPPDE1 overexpression plasmid/NC (negative control) or si-PPPDE1/si-NC. Flow cytometry analysis and CCK-8 assay (used to detect apoptosis and proliferation, respectively), as well as western blotting, showed that the levels of PPPDE1 were positively correlated with the pro-apoptotic molecules Bax and Cyt C. Accordingly, aberrantly high expression of PPPDE1 caused a spatiotemporal imbalance in foetal rats with ARMs during hindgut development. Therefore, the upregulation of PPPDE1 may promote epithelial apoptosis and reduce proliferation in the hindgut via the mitochondrial apoptotic pathway. This could affect the fusion of the URS and cloacal membrane, ultimately inhibiting the hindgut development and resulting in ARMs.

miR-141-3p affects ß-catenin signaling and apoptosis by targeting Ubtd2 in rats with anorectal malformations.

Anorectal malformations (ARMs) are the most common gastrointestinal malformations. miR-141-3p was obtained from whole-transcriptome sequencing, and Ub domain-containing protein 2 (Ubtd2) was predicted as the target gene. An ARM rat model was induced using ethylenethiourea. Fluorescence in situ hybridization and immunofluorescence were used to detect the spatiotemporal expression of miR-141-3p and Ubtd2, respectively. A dual-luciferase reporter assay confirmed their targeting relationship, and cell proliferation and apoptosis were investigated after transfection in the intestinal epithelium (IEC-6). Additionally, western blotting and co-immunoprecipitation were used to examine the protein levels and the endogenous binding relationship. miR-141-3p was downregulated in the ARM group, whereas Ubtd2 increased and colocalized with TUNEL-positive cells. After miR-141-3p inhibition, protein expression of USP5 and ß-catenin was affected via Ubtd2, and USP5 could bind to both Ubtd2 and ß-catenin. Flow cytometry analysis and caspase 3/7 staining demonstrated that downregulated miR-141-3p promoted cell apoptosis through Ubtd2. In summary, targeting Ubtd2 decreased in miR-141-3p and promoted apoptosis of intestinal epithelium and regulated ß-catenin expression. This may cause aberrant apoptosis during hindgut development and mediate the imbalance of ß-catenin signaling in the cloaca, further affecting the occurrence of ARMs.

RNA-Seq Profiling of Circular RNAs During Development of Hindgut in Rat Embryos With Ethylenethiourea-Induced Anorectal Malformations.

Anorectal malformations (ARMs) are among the most common congenital terminal digestive tract malformations. Circular RNAs (circRNAs), a novel type of endogenous non-coding RNAs, play roles in the development of the digestive system; however, their contributions to the pathogenesis of ARMs are not well-established. In this study, we explored the mechanism underlying ethylenethiourea (ETU)-induced ARMs by profiling circRNA expression via RNA-seq and constructing a regulatory circRNA-miRNA-mRNA network. Nine pregnant rats were gavage-fed a single dose of 125 mg/kg 1% ETU (ARM group) on gestational day 10 (GD10), and another 9 pregnant rats received a similar dose of saline (normal group) as a control. Embryos were obtained by cesarean section on the key time-points of anorectal development (GD14, GD15, and GD16). Hindgut samples isolated from the fetuses were evaluated by high-throughput sequencing and differentially expressed circRNAs were validated by reverse transcription-quantitative polymerase chain reaction, agarose gel electrophoresis, and Sanger cloning and sequencing. A total of 18295 circRNAs were identified in the normal and ARM groups. Based on the 425 differentially expressed circRNAs (|Fc| > 2, p < 0.05), circRNA-miRNA and miRNA-mRNA pairs were predicted using miREAP, miRanda, and TargetScan. A total of 55 circRNAs (14 up- and 41 downregulated in the ARM group compared to the normal group) were predicted to bind to 195 miRNAs and 947 mRNAs. Competing endogenous RNA networks and a Kyoto Encyclopedia of Genes and Genomes analysis revealed that novel_circ_001042 had the greatest connectivity and was closely related to ARM-associated signaling pathways, such as the Wingless Type MMTV integration site family, mitogen-activated protein kinase, and transforming growth factor-ß pathways. These results provide original insight into the roles of circRNAs in ARMs and provide a valuable resource for further analyses of molecular mechanisms and signaling networks.

Upregulation of miR-92a-2-5p potentially contribute to anorectal malformations by inhibiting proliferation and enhancing apoptosis via PRKCA/ß-catenin.

Anorectal malformations (ARMs) is one of the most common gastrointestinal anomalies. Previous research revealed that miR-92a-2-5p was upregulated in ARMs. However, the underlying roles remains unknown. The current study was to further investigate the spatiotemporal expression patterns of miR-92a-2-5p and its target gene protein kinase C alpha (PRKCA) predicted by bioinformatic method, and to explore their potential functions in anorectal malformations (ARMs). Rat models with ethylenethiourea-induced ARMs were made for subsequent experiments. Direct target relationship between miR-92a-2-5p and PRKCA was validated using a luciferase reporter assay. The spatiotemporal expression pattern of miR-92a-2-5p was evaluated using fluorescence in situ hybridization (FISH), while the expression of PRKCA was revealed by immunohistochemical staining and western blotting. IEC-6 cells were transfected with mimics/mimics NC (Negative control)/inhibitor/inhibitor NC of miR-92a-2-5p or si-PRKCA/si-PRKCA NC, respectively. Then the downstream molecules of miR-92a-2-5p, PRKCA and ß-catenin, were subsequently detected. Meanwhile, apoptosis and viability assays were measured. Dual luciferase assay confirmed the direct regulatory relationship between miR-92a-2-5p and PRKCA. FISH revealed that miR-92a-2-5p was expressed with a higher level in ARMs fetuses. Further analyses of PRKCA showed lower protein expression level in ARMs group, which was opposite to miR-92a-2-5p. In vitro experiments revealed that overexpression of miR-92a-2-5p or knockdown of PRKCA can down-regulate PRKCA, up-regulate and facilitate nuclear localization of ß-catenin, increase apoptosis and decrease proliferation of IEC-6. Taken together, these findings suggest that aberrantly high expression of miR-92a-2-5p potentially contribute to ARMs by inhibiting proliferation and enhancing apoptosis of intestinal cells via negatively regulating PRKCA/ß-catenin.