Xiaoqun Xu

Circular RNA ZNF609 functions as a competitive endogenous RNA to regulate AKT3 expression by sponging miR-150-5p in Hirschsprung’s disease

Research over the past decade suggested critical roles for circular RNAs in the natural growth and disease progression. However, it remains poorly defined whether the circular RNAs participate in Hirschsprung disease (HSCR). Here, we reported that the cir-ZNF609 was down-regulated in HSCR compared with normal bowel tissues. Furthermore, suppression of cir-ZNF609 inhibited the proliferation and migration of cells. We screened out several putative cir-ZNF609 ceRNAs of which the AKT3 transcript was selected. Finally, RNA immunoprecipitation and luciferase reporter assays demonstrated that cir-ZNF609 may act as a sponge for miR-150-5p to modulate the expression of AKT3. In conclusion, these findings illustrated that cir-ZNF609 took part in the onset of HSCR through the crosstalk with AKT3 by competing for shared miR-150-5p.

SLIT2/ROBO1-miR-218-1-RET/PLAG1: a new disease pathway involved in Hirschsprung's disease

Hirschsprungs disease (HSCR) is a rare congenital disease caused by impaired proliferation and migration of neural crest cells. We investigated changes in expression of microRNAs (miRNAs) and the genes they regulate in tissues of patients with HSCR. Quantitative real‐time PCR and immunoblot analyses were used to measure levels of miRNA, mRNAs, and proteins in colon tissues from 69 patients with HSCR and 49 individuals without HSCR (controls). Direct interactions between miRNAs and specific mRNAs were indentified in vitro, while the function role of miR‐218‐1 was investigated by using miR‐218 transgenic mice. An increased level of miR‐218‐1 correlated with increased levels of SLIT2 and decreased levels of RET and PLAG1 mRNA and protein. The reductions in RET and PLAG1 by miR‐218‐1 reduced proliferation and migration of SH‐SY5Y cells. Overexpression of the secreted form of SLIT2 inhibited cell migration via binding to its receptor ROBO1. Bowel tissues from miR‐218‐1 transgenic mice had nerve fibre hyperplasia and reduced numbers of gangliocytes, compared with wild‐type mice. Altered miR‐218‐1 regulation of SLIT2, RET and PLAG1 might be involved in the pathogenesis of HSCR.

Inhibition of Toll-like receptor 4 with vasoactive intestinal peptide attenuates liver ischemia-reperfusion injury.

BACKGROUND Toll-like receptor 4 (TLR4) has attracted a great deal of attention in ischemia-reperfusion (IR) injury in recent years. Vasoactive intestinal peptide (VIP) plays an important role in anti-inflammatory and immunomodulatory activity in several animal models. There are no data available regarding the effect of VIP on TLR4 expression in IR injury in vivo. In the present study, we study the effect of VIP on TLR4 expression in mouse macrophage cell line RAW 264.7 and a mouse partial IR model. METHODS The potential inhibitory effect of VIP on TLR4 mRNA and protein in a mouse macrophage cell line and in a mouse model of partial warm hepatic IR injury was assessed. We also assessed the expression tumor necrosis factor (TNF)-α and interleukin (IL)-6 in this model. RESULTS Expression of TLR4 mRNA levels was significantly decreased at 6, 12, and 24 hours after treat with VIP in mouse macrophage cell line RAW 264.7. Expression of TLR4 mRNA, TLR4 protein, alanine aminotransferase, TNF-α, and IL-6 levels were significantly increased in the IR group but significantly decreased in groups pretreated with VIP at a concentration of 5 and 10 nmol. Hematoxylin and eosin staining show apparent edema and necrosis were observed in the IR group, but in the VIP pretreatment group, edema and necrosis in IR modes were reduced. CONCLUSION This study showed that VIP might inhibit TLR4 in vitro and in vivo, and pretreatment with VIP might inhibited TLR4 activation and reduced warm IR injury.

Aberrant reduction of MiR-141 increased CD47/CUL3 in Hirschsprung's disease.

Background: MiR-141 has been confirmed to be associated with various human diseases. However, whether miR-141 is involved in the pathogenesis of Hirschsprungs disease (HSCR) remains unknown. Here, we design the experiment to reveal the relationship between miR-141 and HSCR. Methods: Quantitative real-time PCR and Western blot were used to detect the expression levels of miR-141 and its potential genes in 70 tissues of HSCR compared with 60 controls. Bisulfite sequencing PCR (BSP) assay was applied to explain the possible mechanism of the aberrant expression level of miR-141. We employed a dual-luciferase reporter assay to validate the regulation relation between miR-141 and CD47/CUL3. Cell migration, proliferation, apoptosis, and cell cycle progression were examined by transwell assay, MTT assay, and flow cytometry, respectively. Results: MiR-141 was down-regulated whereas CD47 and CUL3 expression was increased in colon tissues from patients with HSCR compared with control group, The increased level of CD47 and CUL3 induced by miR-141 reduced proliferation and migration of 293T and SH-SY5Y cells. Furthermore, this suppression was reversed by reducing of CD47 and CUL3. Hypermethylation of a CpG Island in the promoter region of miR-141 gene was confirmed in HSCR tissues. Conclusion: Aberrant reduction of miR-141 may play an important role in the pathogenesis of HSCR with the inhibiting affection on cell migration and proliferation abilities. The present study demonstrates for the first time the role of miR-141 and its target genes in the occurrence of HSCR, and provides us a new direction for the study of the pathogenesis of Hirschsprungs disease.

Specific serum microRNA profile in the molecular diagnosis of Hirschsprung's disease

Hirschsprungs disease (HSCR), a congenital gastrointestinal disorder, is one of the most common causes of neonatal bowel obstruction. Without an early screening and diagnosis, some patients develop serious complications, such as toxic megacolon or acute enterocolitis. We sought to identify specific serum microRNAs (miRNAs) that can serve as novel early, non‐invasive screening signature and then to test their specificity and sensitivity in diagnosing Hirschsprungs disease. We obtained serum samples from 95 HSCR cases and 104 matched controls. An initial screening of miRNA expression was performed through TaqMan Low Density Array. The candidate miRNAs were validated by individual reverse transcription quantitative real‐time PCR arranged in the training and a two‐stage validation set. Additional double‐blind testing was performed in 23 patients with clinically suspected HSCR to evaluate the diagnostic value and accuracy of the serum miRNA profile in predicting HSCR. Following a multi‐stage evaluation approach, five miRNAs were significantly increased in HSCR cases compared with controls. The areas under the receiver operating characteristic (ROC) curve of this five‐serum miRNA signature were 0.895, 0.893 and 0.925 in training set and two validation sets, respectively. The accuracy rate of the five‐miRNA profile as HSCR signature was 82.6%, which, in the double‐blind testing set, was markedly higher than that of contrast enema (70%), the most commonly used test performed to diagnose HSCR. Our results indicate that a five‐serum miRNA signature may be linked to HSCR, representing a potential, novel, non‐invasive diagnostic approach for early screening of HSCR.

Down-Regulation of MicroRNA-146a in the Early Stage of Liver Ischemia-Reperfusion Injury

BACKGROUND MicroRNAs (miRNAs), 21-23-nucleotide noncoding RNAs, act as regulators of gene expression transcriptionally. MicroRNA-146a(miR-146a) has been demonstrated to be one of the key molecules in oncogenesis and inflammatory responses. Few data describe the expression of miR-146a in liver ischemia-reperfusion (IR) injury. The present study sought to explore the relationship of miR-146a to Toll-like receptor 4 (TLR4) signaling pathways in a rat model of warm IR injury. METHODS The expression of miR-146a was detected by real-time reverse-transcriptase polymerase chain reaction using a partial warm hepatic IR injury model. The expression of TLR4, tumor necrosis factor receptor-associated factor 6 (TRAF6), and interleukin-1 receptor-associated kinase (IRAK 1) protein was assessed by Western blotting as well as the signaling pathways induced by TLR4. RESULTS The expression of hepatic miR-146a was down-regulated in IR injury during the 24 hours after reperfusion, reaching the lowest level at 6 hours after reperfusion. Increases in TLR4, TRAF6, and IRAK1 were accompanied by decreased miR-146a during the 24 hours after reperfusion, peaking at 6 hours. Immunohistochemistry showed cytoplasmic expression of cells positive for TLR4, and nuclear expression of cells positive for nuclear factor κB p65 and c-jun to be increased among IR groups after reperfusion. CONCLUSION miR-146a was down-regulated in the early stage of liver IR injury.

Use of small intestinal submucosal and acellular dermal matrix grafts in giant omphaloceles in neonates and a rabbit abdominal wall defect model.

BACKGROUND The described surgical strategies for the management of omphalocele include primary closure, staged closure, and delayed closure. A primary repair is not suitable for all giant omphaloceles. We implanted two grafts, small intestinal submucosal (SIS) and acellular dermal matrix (ADM) onto abdominal wall defects in neonates to study the safety and efficacy of SIS and ADM graft techniques for initial closure of giant omphaloceles in infants, and we also implanted these grafts onto abdominal wall defects in an animal model. METHODS Twenty-four patients with giant omphaloceles were divided into two groups (ADM group, 12 patients; SIS group, 12 patients). The operative time, skin healing time postoperatively, and the incidence of skin infections, and abdominal wall hernias were observed. In the rabbit animal model, bilateral full-thickness incisions were made through the rabbit rectus abdominus muscles and a 2×4cm longitudinal whole layer defect was created on either the left or right lateral anterior abdominal wall. A four-layered variant of the SIS graft was used to repair the right abdominal defect; ADM was used to repair the left. Tensile strength was measured using an Instron tensiometer. Electron scanning and light microscopy were used to evaluate neovascularization, collagen deposition, and muscle fibers at 2, 4, 8, and 16weeks postimplantation. RESULTS In the neonatal patients, there was no statistically significant difference between the two groups with respect to operative time, skin healing time postoperatively, the incidence of skin infections, or abdominal wall hernias. In the SIS group, only one patient developed a skin infection, which led to skin necrosis and sloughing. In the ADM group, four patients developed skin infection postoperatively, and the patch was gradually removed. In the animal study, there was no significant difference between the mean breaking strength of ADM versus SIS repairs. Scanning electron and light microscopy showed collagen deposition, increased vascularization, fibroblasts, and muscular regeneration in both SIS and ADM repairs. SEM showed that the SIS graft was absorbed, while ADM was not. Light microscopy showed foreign body macrophages in ADM, but not in the SIS repairs. CONCLUSION SIS and ADM grafts adequately enhance healing with a low complication rate. Compared with ADM grafts, SIS is absorbable, induces less inflammation, and is more biocompatible, and therefore might be more useful and suitable for closure of abdominal wall defects.

Aberrant high expression of NRG1 gene in Hirschsprung disease.

BACKGROUND/PURPOSE Hirschsprung disease (HSCR) is a congenital disorder characterized by the absence of intramural ganglion cells along with variable lengths of the gastrointestinal tract. Recent studies have indicated the potential function of neuregulin-1 (NRG1) in HSCR, which encodes the heregulins and other mitogenic ligands for the ErbB family. The purpose of this study was to further clarify the role of NRG1 in the pathogenesis of HSCR. METHODS We examined the NRG1 messenger RNA (messenger RNA) and protein expression levels in gut tissues of 63 patients with sporadic HSCR (both stenotic and dilated gut tissues) and 35 controls. Moreover, using the methylation-specific polymerase chain reaction, we examined the methylation pattern of exon 1 of the NRG1 gene. RESULTS The mRNA expression levels of NRG1 were significantly higher in tissues of HSCR than those in controls, and the increased NRG1 protein levels in HSCR were consistent with the mRNA levels. However, no methylation pattern change was observed in exon 1 of the gene among different groups. CONCLUSIONS Our study demonstrates that the aberrant expression of NRG1 may play an important role in the pathology of HSCR. DNA methylation of the gene seems not to be involved in the mechanism of such aberrant expression, and other factors should be explored.

Exome-Wide Association Study Identified New Risk Loci for Hirschsprung’s Disease

Hirschsprung disease (HSCR) is a rare congenital disease caused by impaired proliferation and migration of neural crest cells. In this study, we aimed to investigate the genetic loci involved in the pathogenesis of HSCR. The exome-wide scan was performed to screen the genetic variants with minor allele frequency (MAF) < 0.05 in exonic regions. Candidate mutation type and the wild type were overexpressed to investigate the affection on cell proliferation and migration. We found that ten variants were associated with HSCR at P < 10−4 in the single-variant analysis while ten genes were also associated with HSCR at P < 10−4 in the optimized sequence kernel association test (SKAT-O) test analysis. Among these SNPs, the missense variants catechol-O-methyltransferase (COMT) (rs6267) and armadillo repeat gene deleted in velocardiofacial syndrome (ARVCF) (rs80068543) indicated an ectopic expression in colon tissues of HSCR patients. The Ala72Ser variant in COMT induced proliferation suppression through NOTCH signal pathway, while the ARVCF affected cell migration via the downregulating of RHOA and ROC. In conclusion, this exome array study identified the COMT and ARVCF missense coding variants as candidate loci for HSCR. The finding implies the abnormal variant of COMT and ARVCF may account for the pathogenesis of HSCR.

Single-stage transanal endorectal pull-through procedure for correction of Hirschsprung disease in neonates and nonneonates: A multicenter study

PURPOSE The outcomes of single-stage transanal endorectal pull-through (SSTEPT) for Hirschsprung disease (HSCR) in young patients are favorable; however, reports have shown that diagnosis and surgery at young ages increase the risk for postoperative enterocolitis and slows postoperative recovery. The present study was primarily designed to evaluate the outcomes of SSTEPT in a multi-institutional cohort of neonates and nonneonates with HSCR. METHODS Between August 2005 and May 2012, a total of 650 children with HSCR were divided into the following two groups: group A (neonatal group, operative age<28days [n=186]); and group B (nonneonatal group, operative age>28days [n=464]). The short-term outcomes were postoperative enterocolitis, perianal excoriation, and anastomotic stricture and leakage rates. The midterm outcomes were incomplete continence and constipation rates based on multi-institutional chart review. Statistical analyses were performed using chi-square (χ2) tests. RESULTS Follow-up was completed in 112 neonates and 303 nonneonates. Short-term outcomes indicated a higher incidence of perianal excoriation (27.6% vs. 6.6%, χ2=33.70, p<0.05), anastomotic strictures (14.3% vs. 6.0%, χ2=27.18, p<0.05), anastomotic leakage (8.0% vs. 1.7%, χ2=8.36, p<0.05), and postoperative enterocolitis (40.2% vs. 10.2%, χ2=49.05, p<0.05) in group A compared to group B. Midterm outcomes indicated a higher incidence of incomplete continence (35.7% vs. 14.9%, χ2=21.85, p<0.05) in group A compared to group B. CONCLUSION Performing single-stage transanal endorectal pull-through in the nonneonatal period may be more appropriate than the neonatal period. There were higher rates of perianal excoriation, anastomotic strictures and leakage, postoperative enterocolitis, and incomplete continence postoperatively in neonates than nonneonates.