Spatial transcriptomics reveals gene interactions and signaling pathway dynamics in rat embryos with anorectal malformation.

Anorectal malformation (ARM) is a prevalent early pregnancy digestive tract anomaly. The intricate anatomy of the embryonic cloaca region makes it challenging for traditional high-throughput sequencing methods to capture location-specific information. Spatial transcriptomics was used to sequence libraries of frozen sections from embryonic rats at gestational days (GD) 14 to 16, covering both normal and ARM cases. Bioinformatics analyses and predictions were performed using methods such as WGCNA, GSEA, and PROGENy. Immunofluorescence staining was used to verify gene expression levels. Gene expression data was obtained with anatomical annotations of clusters, focusing on the cloaca region's location-specific traits. WGCNA revealed gene modules linked to normal and ARM cloacal anatomy development, with cooperation between modules on GD14 and GD15. Differential gene expression profiles and functional enrichment were presented. Notably, protein levels of Pcsk9, Hmgb2, and Sod1 were found to be downregulated in the GD15 ARM hindgut. The PROGENy algorithm predicted the activity and interplay of common signaling pathways in embryonic sections, highlighting their synergistic and complementary effects. A competing endogenous RNA (ceRNA) regulatory network was constructed from whole transcriptome data. Spatial transcriptomics provided location-specific cloaca region gene expression. Diverse bioinformatics analyses deepened our understanding of ARM's molecular interactions, guiding future research and providing insights into gene regulation in ARM development.

Rack1-mediated ferroptosis affects hindgut development in rats with anorectal malformations: Spatial transcriptome insights.

Anorectal malformation (ARM), a common congenital anomaly of the digestive tract, is a result of insufficient elongation of the urorectal septum. The cytoplasmic protein Receptor of Activated C-Kinase 1 (Rack1) is involved in embryonic neural development; however, its role in embryonic digestive tract development and ARM formation is unexplored. Our study explored the hindgut development and cell death mechanisms in ARM-affected rats using spatial transcriptome analysis. We induced ARM in rats by administering ethylenethiourea via gavage on gestational day (GD) 10. On GDs 14-16, embryos from both normal and ARM groups underwent spatial transcriptome sequencing, which identified key genes and signalling pathways. Rack1 exhibited significant interactions among differentially expressed genes on GDs 15 and 16. Reduced Rack1 expression in the ARM-affected hindgut, verified by Rack1 silencing in intestinal epithelial cells, led to increased P38 phosphorylation and activation of the MAPK signalling pathway. The suppression of this pathway downregulated Nqo1 and Gpx4 expression, resulting in elevated intracellular levels of ferrous ions, reactive oxygen species (ROS) and lipid peroxides. Downregulation of Gpx4 expression in the ARM hindgut, coupled with Rack1 co-localisation and consistent mitochondrial morphology, indicated ferroptosis. In summary, Rack1, acting as a hub gene, modulates ferrous ions, lipid peroxides, and ROS via the P38-MAPK/Nqo1/Gpx4 axis. This modulation induces ferroptosis in intestinal epithelial cells, potentially influencing hindgut development during ARM onset.

Is enema reduction in pediatric intussusception with a history of over 48 h safe: A retrospective cohort study.

BACKGROUND: Intussusception is one of the most common acute abdominal diseases in children. Enema reduction is the first-line treatment for intussusception in good condition. Clinically, a history of disease over 48 h is usually listed as a contraindication for enema reduction. However, with the development of clinical experience and therapy, an increasing number of cases have shown that the prolongation of the clinical course of intussusception in children is not an absolute contraindication for enema treatment. This study aimed to analyze the safety and efficacy of enema reduction in children with a history of disease longer than 48 h. METHODS: We conducted a retrospective matched-pair cohort study of pediatric patients with acute intussusception between 2017 and 2021. All patients were treated with ultrasound-guided hydrostatic enema reduction. According to the length of history, the cases were classified into two groups: history <48 h (<48 h group) and history greater than or equal to 48 h (≧48 h group). We generated a 1:1 matched-pair cohort matched for sex, age, admission time, main symptoms, and concentric circle size on ultrasound. Clinical outcomes were compared between the two groups, including success, recurrence, and perforation rates. RESULTS: From January 2016 to November 2021, 2701 patients with intussusception were admitted to the Shengjing Hospital of China Medical University. A total of 494 cases were included in the ≧48 h group, and 494 cases with a history of <48 h were selected for matched comparison in the <48 h group. The success rates of the ≧48 h and <48 h groups were 98.18% vs. 97.37% (p = 0.388), and the recurrence rates were 13.36% vs. 11.94% (p = 0.635), showing no difference according to the length of history. The perforation rate was 0.61% vs. 0%, respectively, with no significant difference (p = 0.247).The comparison of the different history groups showed that in patients with bloody stools, the length of history had no significant effect on the enema reduction outcome(94.90% vs. 86.76%, p = 0.064). CONCLUSIONS: Ultrasound-guided hydrostatic enema reduction is safe and effective for pediatric idiopathic intussusception with a history of ≧48 h.

CircJag1 promotes apoptosis of ethylene thiourea-exposed anorectal malformations through sponging miR-137-3p by regulating Sox9 and suppressing Wnt/ß-catenin pathway during the hindgut development of rat embryos.

Anorectal malformations (ARMs) are common birth defects involving congenital structural anomalies of the gastrointestinal tract. As an important component of non-coding RNAs, circular RNAs (circRNAs) widely participate in the digestive system development; however, the specific molecular mechanism of their involvement in ARM occurrence remains obscure. Herein, we generated rat models of ARMs induced by ethylene thiourea. A novel circRNA (circJag1) was screened and identified by RNA-Seq, which is remarkably upregulated in hindgut tissues of ARM rat embryos. In vivo experiments, colocation analysis via fluorescence in situ hybridization, and immunofluorescence further demonstrated that the disordered circJag1/miR-137-3p/Sox9 expression caused a spatiotemporal imbalance in the urorectal septum (URS) of ARMs. In vitro, functional assays confirmed that circJag1 upregulation resulted in the degradation of nuclear ß-catenin, C-myc, and Cyclin D1 in rat intestinal epithelial cells, as well as the promotion of apoptosis and suppression of cell proliferation. Mechanistically, dual-luciferase reporter assay and RNA immunoprecipitation assay indicated that circJag1 acted as a miR-137-3p sponge, thereby inhibiting its repressive effect on its target Sox9. Further experiments showed that a loss of Sox9 abolished the circJag1-mediated increase in apoptosis. In conclusion, aberrantly high circJag1 expression promotes epithelial apoptosis by suppressing the canonical Wnt/ß-catenin pathway via the miR-137-3p/Sox9 axis, which leads to fusion failure of the URS and cloacal membrane, and eventually contributed to ARMs. Our achievements might boost the comprehension of ARM pathogenesis and could provide a novel candidate target for the development of therapies for ARMs to complement surgical treatment.

Radiological and clinical characteristics of intussuscepted, inverting, and inverted Meckel's diverticulum: A case series.

PURPOSE: Inverted Meckel's diverticulum (IMD) is a well-established but rare disease. This study aimed to summarize the radiological and clinical characteristics of IMD, and correlates its radiological and surgical findings to obtain an accurate early preoperative diagnosis. METHOD: This is a retrospective study included IMD patients from a large children's medical center in China, between January 2009 and March 2022. We reviewed demographic data, clinical manifestations, preoperative examinations, surgical findings, histopathological results, and outcomes. RESULTS: Twenty-three cases with IMD (14 male patients [60.9%]; median age, 6.7 years; age range, 9 months to 13 years) were retrospectively reviewed over a period of 13 years. The typical clinical manifestations of IMD included abdominal pain, vomiting, and abdominal tenderness. The most commonly used imaging modalities were abdominal ultrasound and computed tomography. This is the first case series on pediatric IMD, that describes the clinical process of IMD, proposes clinical phases of IMD, and first summarizes the radiological findings characteristic of each clinical phase. CONCLUSIONS: The clinical process of IMD can be divided into four phases (intussuscepted Meckel's diverticulum [MD], inverting MD, inverted MD, intussusception secondary to IMD). Patients in different clinical phases present with various radiological features. Mastering the radiological and clinical characteristics of each phase of IMD can aid in its early diagnosis and timely operative intervention, thus avoiding unnecessary intestinal necrosis and resection.

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.

Diagnosis and management of fecal incontinence in children and adolescents.

Fecal incontinence (FI) is a commonly occurring disease of high concern. It is characterized by voluntary and involuntary defecation in children and adolescents. It is not only a physical disease but also a psychological and behavioral disorder. FI poses a serious burden on individuals and their families and therefore has become a social problem. Unfortunately, the management of FI among children is still a challenge because the etiology varies widely. Constipation has been found to be the most common cause, while sphincter dysfunction and neurogenic abnormalities may also play a role. Currently, no consensus guidelines exist, and the criteria for selecting optional methods remain unclear. It is therefore necessary to improve the efficacy of diagnosis and management strategies of FI in children. This review focused on the classification and etiology, discussed the diagnosis and management methods of FI in children and adolescents, and aimed to guide future studies.

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.

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.

Superficial CD34-Positive Fibroblastic Tumor on the Chest Wall of an 8-Year-Old Girl: A Case Report and Literature Review.

This study reports a case of superficial CD34-positive fibroblastic tumor (SCPFT) in a child and analyze the major known clinicopathological features of SCPFT and other skin mesenchymal tumors, contributing to an accurate diagnosis of this rare disease. We summarize the clinicopathologic features of an 8-year-old girl who was diagnosed with SCPFT and 46 previously reported SCPFT cases. Post-operative histopathologic examination of the current case showed the tumor lesion was well-circumscribed; tumor cells were spindled-to-polygonal with a fascicular pattern; most nuclei displayed hyperchromasia and low mitotic rate; intranuclear pseudoinclusions could be found; and abundant eosinophilic cytoplasm and partial myxoid stroma were observed. Immunohistochemistry revealed strong and diffuse CD34-positivity, vimentin staining positively but no S-100, SMA, NSE, CD31, desmin, cytokeratin, STAT6, ß-catenin, MDM2, or ERG expression. The Ki-67 and CD68 labeling indexes were approximately 1%. There were no rearrangements of PDGFB or PRDM10 tested by FISH. After surgical resection, the patient had no signs of recurrence or metastasis at a 6-month follow-up. The present case is the first that describes SCPFT in children and has significant clinical implications. SCPFT should be differentiated from other skin mesenchymal tumors. The presented compilation of all so far published SCPFT cases will help in diagnosing successfully SCPFT and increasing awareness of this tumor to guide clinical practice.

Ultrasound-guided hydrostatic reduction versus fluoroscopy-guided air reduction for pediatric intussusception: a multi-center, prospective, cohort study.

BACKGROUND: Intussusception is the most common abdominal emergency in children. The first line treatment of uncomplicated pediatric intussusception is enema reduction. Until now, there have been no multi-center studies comparing the effectiveness and safety of UGHR and FGAR in the treatment of pediatric intussusception. The aim of this study was to compare the effectiveness and safety of the two most commonly used enema methods of pediatric intussusception: ultrasound-guided hydrostatic reduction (UGHR) and fluoroscopy-guided air reduction (FGAR). METHODS: From November 1, 2017 to October 31, 2018, we conducted a multi-center, prospective, cohort study. Children diagnosed with intussusception in four large Children's Medical Centers in China were divided into UGHR and FGAR groups. Stratified analysis and subgroup analysis were used for further comparison. The success and recurrence rates were used to evaluate the effectiveness of enema reduction. The perforation rate was used to evaluate the safety of enema reduction. RESULTS: A total of 2124 cases met the inclusion criteria (UGHR group: 1119 cases; FGAR group: 1005 cases). The success and recurrence rates in the UGHR group were higher than in the FGAR group (95.80%, 9.28% vs. 93.13%, 10.65%) (P < 0.05, P > 0.05), respectively. The perforation rate in the UGHR group was 0.36% compared with 0.30% in the FGAR group (P > 0.05). Subgroup analysis showed the success rates in the UGHR group were higher than in the FGAR group of patients with onset time between 12 and 24 h (95.56% vs. 90.57%) (P < 0.05). Of patients aged 4 to 24 months, the success rates in the UGHR group were also higher than in the FGAR group (95.77% vs. 91.60%) (P < 0.05). Stratified analysis showed the success rates in the UGHR group were higher than in the FGAR group in patients with the symptom of bloody stool (91.91% vs 85.38%) (P < 0.05). CONCLUSIONS: UGHR and FGAR are safe, nonsurgical treatment methods for acute pediatric intussusception. UGHR is superior to FGAR, no radiation risk, its success rate is higher, without a difference in perforation rate, especially for patients aged 4-24 months. LEVEL OF EVIDENCE: Level II.

Association of air temperature with pediatric intussusception in northeastern China: A 10-year retrospective study.

OBJECTIVE: The aim of this study was to determine whether an association existed between intussusception and air temperature. METHODS: A retrospective study was performed between March 2006 and February 2016 to determine the relationship between pediatric primary intussusception (PPI) and air temperature. Information from hospital records of 5922 cases of PPI and Mean daily temperatures of Shenyang were obtained. Pearson correlation analysis was used to examine the association between monthly PPI cases and monthly mean temperature. Factorial analysis-of-variance was used to examine differences in the numbers of seasonal PPI cases during different seasons. RESULTS: Monthly PPI cases fluctuated throughout the year, with a peak in June, and a trough in February. Pearson correlation analysis showed that monthly PPI cases was associated with the monthly mean temperature (p < 0.01). Factorial analysis-of-variance showed there was significant difference in the numbers of seasonal PPI cases during different seasons. Multiple comparison showed a significant difference in seasonal PPI cases between spring and summer, spring and winter, summer and autumn, summer and winter, autumn and winter (p < 0.01). CONCLUSIONS: Monthly PPI cases were positively associated with monthly mean temperature in Shenyang. The incidence of intussusception shows a seasonal trend, with a peak in summer (May to July).

Sox10 Is a Specific Biomarker for Neural Crest Stem Cells in Immunohistochemical Staining in Wistar Rats.

OBJECTIVE: Neural crest stem cells (NCSCs) are prototypically migratory cells immigrating from the dorsal neural tube to specific embryonic sites where they generate a variety of cell types. A lot of biomarkers for NCSCs have been identified. However, which biomarkers are the most specific is still unclear. METHODS: The rat embryos harvested in embryonic day 9 (E9), E9.5, E10, E10.5, E11, E12, E13, and E14 were paraffin-embedded and sectioned in transverse. NCSCs were spatiotemporally demonstrated by immunohistochemical staining with RET, p75NTR, Pax7, and Sox10. NCSCs were isolated, cultured, and stained with RET, p75NTR, Pax7, and Sox10. RESULTS: In the paraffin sections of rat embryos, the immunohistochemical staining of RET, p75NTR, and Sox10 can all be used in demonstrating NCSCs. Sox10 was positive mainly in NCSCs while RET and p75NTR were positive not only in NCSCs but also in other tissue cells. In primary culture cells, Sox10 was mainly in the nucleus of NCSCs, RET was mainly in the membrane, and p75NTR was positive in cytoplasm and membrane. CONCLUSIONS: Sox10 is the specific marker for immunohistochemical staining of NCSCs in paraffin sections. In cultured cells, Sox10, p75NTR, and RET presented a similar staining effect.

Gastroesophageal Reflux Poses a Potential Risk for Late Complications of Bronchopulmonary Dysplasia: A Prospective Cohort Study.

BACKGROUND: Bronchopulmonary dysplasia (BPD) is the most common respiratory disorder in extremely low birth weight infants. Although most symptoms of BPD improve, some late complications exist, even with regular treatment. Gastroesophageal reflux (GER), also common in extremely premature infants, may be related to many cardiorespiratory symptoms. However, the potential of GER as a risk factor for late complications associated with BPD is still unclear. RESEARCH QUESTION: The goal of this study was to determine if GER increases the risk of late complications of BPD in infants. STUDY DESIGN AND METHODS: A multicenter prospective cohort of 131 infants (79 male subjects, 52 female subjects) with BPD was enrolled. The development of late complications was assessed over an 18-month follow-up period. Twenty-four-hour pH-multichannel intraluminal impedance and gastric sodium concentrations were analyzed in all infants at 36 weeks' postmenstrual age and at the last interview. Prevalence and risk factors of late complications of BPD were analyzed by using forward logistic regression. RESULTS: The prevalence of late complications in BPD infants was 63.79% and included respiratory symptoms (49.14%), vomiting (38.79%), retinopathy of prematurity (25.86%), hypoxic-ischemic injury (3.45%), rehospitalization (26.72%), and sudden death (0.86%). Respiratory diseases constituted the most frequent complication. The prevalence of GER in BPD was 42.24% and included acid GER (18.10%) and duodenogastroesophageal reflux (DGER; 24.14%). Risk factors for respiratory symptoms were gestational age ≤ 30 weeks (OR, 3.213; 95% CI, 1.221-8.460), birth weight < 1,500 g (OR, 2.803; 95% CI, 1.014-7.749), invasive ventilation > 7 days (OR, 4.952; 95% CI, 1.508-16.267), acid GER (OR, 4.630; 95% CI, 1.305-16.420), and DGER (OR, 5.588; 95% CI, 1.770-17.648). Infants with BPD and DGER were more prone to late complications than those with acid GER or no reflux. INTERPRETATION: The prevalence of late complications is high in infants with BPD. GER (and in particular, DGER) poses a tentative risk for these late complications. TRIAL REGISTRY: ClinicalTrials.gov; No.: NCT03014453; URL: www.clinicaltrials.gov.

miR-322 treatment rescues cell apoptosis and neural tube defect formation through silencing NADPH oxidase 4.

AIMS: Failure of neural tube closure resulting from excessive apoptosis leads to neural tube defects (NTDs). NADPH oxidase 4 (NOX4) is a critical mediator of cell growth and death, yet its role in NTDs has never been characterized. NOX4 is a potential target of miR-322, and we have previously demonstrated that miR-322 was involved in high glucose-induced NTDs. In this study, we investigated the effect of NOX4 on the embryonic neuroepithelium in NTDs and reveal a new regulatory mechanism for miR-322 that disrupts neurulation by ameliorating cell apoptosis. METHODS: All-trans-retinoic acid (ATRA)-induced mouse model was utilized to study NTDs. RNA pull-down and dual-luciferase reporter assays were used to confirm the interaction between NOX4 and miR-322. In mouse neural stem cells and whole-embryo culture, Western blot and TUNEL were carried out to investigate the effects of miR-322 and NOX4 on neuroepithelium apoptosis in NTD formation. RESULTS: NOX4, as a novel target of miR-322, was upregulated in ATRA-induced mouse model of NTDs. In mouse neural stem cells, the expression of NOX4 was inhibited by miR-322; still further, NOX4-triggered apoptosis was also suppressed by miR-322. Moreover, in whole-embryo culture, injection of the miR-322 mimic into the amniotic cavity attenuated cell apoptosis in NTD formation by silencing NOX4. CONCLUSION: miR-322/NOX4 plays a crucial role in apoptosis-induced NTD formation, which may provide a new understanding of the mechanism of embryonic NTDs and a basis for potential therapeutic target against NTDs.

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.

Status survey on enema reduction of paediatric intussusception in China.

OBJECTIVE: Intussusception is a common paediatric abdominal emergency in infants. The first-line treatment of choice in uncomplicated paediatric intussusception is enema reduction. The study aim was to provide an overview of the current national practice of enema reduction of paediatric intussusception in China. METHODS: A questionnaire on enema reduction of paediatric intussusception was sent to respondents (members of the Pediatric Anorectal Group, the Neonatal Group, the Society of Pediatric Surgery and the China Medical Association). RESULTS: Data from 128 questionnaires were analysed. Of these, 78.1% (100/128) reported the use of fluoroscopy, 17.2% (22/128) use of ultrasound monitoring, 78.9% (101/128) use of air and 17.9% (23/128) use of normal saline. A total of 78.9% (101/128) reported a success rate of 90%, 25.8% (33/128) reported that a paediatric surgeon managed the reduction, 18.8% (24/128) that a radiologist managed the reduction and 44.5% (57/128) that a paediatric surgeon and radiologist jointly managed the reduction. CONCLUSIONS: There is large variation in the techniques of enema reduction of intussusception in China. Fluoroscopy-guided air enema reduction is mainly used. Enema reduction of uncomplicated cases of paediatric intussusception in China lacks standardization of equipment and personnel involvement.

Mesenteric heterotopic pancreas in a pediatric patient: A case report and review of literature.

Heterotopic pancreas (HP) is a congenital anomaly defined as pancreatic tissue that has no contact with the orthotopic pancreas and its own duct system and vascular supply. The most common locations of HP are the upper gastrointestinal tract, specifically, the stomach, duodenum, and proximal jejunum. Involvement of the mesentery is rare. Here, we describe a rare case of mesenteric heterotopic pancreas (MHP) in a 12-year-old girl who presented with acute abdomen. The patient underwent emergency laparotomy, and the mass and adjacent small bowel were resected. Results of the postoperative histopathologic examination confirmed the diagnosis of MHP. Observation of the patient for 12 mo postoperatively showed no evidence of recurrence. Preoperative diagnosis of HP is difficult, even in a symptomatic patient. Increased awareness and understanding of the image characteristics of MHP will aid in correct preoperative diagnosis and appropriate patient management.

Microarray analysis of miRNAs during hindgut development in rat embryos with ethylenethiourea­induced 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 ETU­treated and control groups. The rats in the ETU­treated group were gavage­fed 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 transcription­quantitative polymerase chain reaction (RT­qPCR) 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 |log2(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 ARM­associated signaling pathways. The RT­qPCR analysis revealed that three miRNA (miR), miR­125b­2­3p, miR­92a­2­5p and miR­99a­5p, were significantly differentially expressed in rats with ARMs compared with the normal group. In conclusion, the results suggested that the differential expression of miR­125b­2­3p, miR­92a­2­5p and miR­99a­5p during key time-points of anorectal formation in rats may have functions in the pathogenesis of ARM.

Expression pattern of Wif1 and ß-catenin during development of anorectum in fetal rats with anorectal malformations.

PURPOSE: This study was performed to investigate the expression pattern of Wnt inhibitory factor 1 (Wif1) and ß-catenin during anorectal development in normal and anorectal malformation (ARM) embryos and the possible role of Wif1 and ß-catenin in the pathogenesis of ARM. METHODS: ARM was induced with ethylenethiourea on the 10th gestational day in rat embryos. Cesarean deliveries were performed to harvest the embryos. The expression pattern of Wif1 and ß-catenin protein and mRNA was evaluated in normal rat embryos (n = 288) and ARM rat embryos (n = 306) from GD13 to GD16 using immunohistochemical staining, Western blot, and real time RT-PCR. RESULTS: Immunohistochemical staining revealed that in normal embryos Wif1 was constantly expressed in the cloaca from GD13 to GD16. On GD13 and GD14, Wif1-immunopositive cells were extensively expressed in the cloaca. On GD15, the expression of Wif1 were mainly detected on the very thin anal membrane. In ARM embryos, the epithelium of the hindgut and urorectal septum demonstrated faint immunostaining for Wif1 from GD14 to GD16. Western blot and real time RT-PCR revealed that Wif1 and ß-catenin protein and mRNA expression level was significantly decreased in the ARM groups compared with the normal group on GD14 and GD15 (p < 0.05). CONCLUSIONS: This study demonstrated that the expression pattern of Wif1 and ß-catenin was disrupted in ARM embryos during anorectal morphogenesis, which demonstrated that downregulation of Wif1 and ß-catenin at the time of cloacal separation into the primitive rectum and urogenital septum might related to the development of ARM.