Weibing Tang

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.

Nidogen‐1 is a common target of microRNAs MiR‐192/215 in the pathogenesis of Hirschsprung's disease

Recent studies have emphasized the important role of microRNA (miRNA) clusters and common target genes in disease progression. Despite the known involvement of the miR‐192/215 family in many human diseases, its biological role in Hirschsprung disease (HSCR) remains undefined. In this study, we explored the role of the miR‐192/215 family in the pathogenesis of HSCR. Quantitative real‐time PCR and western blotting measured relative expression levels of miRNAs, mRNAs, and proteins in 80 HSCR patients and 77 normal colon tissues. Targets were evaluated by dual‐luciferase reporter assays, and the functional effects of miR‐192/215 on human 293T and SH‐SY5Y cells were detected by the Transwell assay, CCK8 assay and flow cytometry. MiR‐192/215 was significantly down‐regulated in HSCR tissue samples, and their knockdown inhibited cell migration and proliferation in the human 293T and SH‐SY5Y cell lines. Nidogen 1 (NID1) was confirmed as a common target gene of miR‐192/215 by dual‐luciferase reporter gene assay and its expression was inversely correlated with that of miR‐192/215 in tissue samples and cell lines. Silencing of NID1 could rescue the extent of the suppressing effects by miR‐192/215 inhibitor. The down‐regulation of miR‐192/215 may contribute to HSCR development by targeting NID1. We proposed the following cascade for the proposed mechanism of miR‐192/215 in the pathogenesis of Hirschsprung disease (HSCR) by targeting Nidogen 1 (NID1). Aberrant expression of miR‐192/215 inhibits cell migration and cell proliferation via NID1. We think the miR‐192/miR‐215/NID1 signaling pathway may play an important role in the pathogenesis of HSCR.

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.

MiR-195 affects cell migration and cell proliferation by down-regulating DIEXF in Hirschsprung’s Disease

BackgroundHirschsprung’s disease (HSCR) is the most common congenital gut motility disorder. We aimed to investigate the roles of miR-195 in the pathogenesis of HSCR.MethodsIn this study, we measured the expression levels of miRNA, mRNA, and protein in colon tissues from 78 patients with HSCR and 66 controls without HSCR. Transwell, Cell Counting Kit-8 (CCK-8) and flow cytometry assay were employed to detect the function role of miR-195 in vitro.ResultsOur results showed that expression levels of miR-195 from patients with HSCR were significantly higher than control group; along with aberrant lower expression levels of digestive-organ expansion factor (DIEXF) were tested. Increased level of miR-195 could suppress the level of DIEXF in cell, which induced the impairment of cell migration and proliferation.ConclusionsAberrant expression of miR-195 may involved in the pathogenesis of HSCR by down-regulated the level of DIEXF.

The relationship between prenatal exposure to BP-3 and Hirschsprung's disease

Hirschsprungs disease (HSCR) is neonatal intestinal abnormality which derived from the faliure of enteric neural crest cells migration to hindgut during embryogenesis from 5 to 12 weeks. Currenly, the knowledge of environmental factors contributing to HSCR is still scarce. Benzophenone-3 (BP-3) is one of the most widely used UV filters, and has weak estrogen and strong anti-androgenic effects. In order to examine the effect of maternal BP-3 exposure on development of offspring and explore the potential mechanism, we conducted case and control study and in vitro study. In this work, BP-3 concertrations in maternal urine was detected by ultra-high performance liquid chromatography. Besides, we investigated the cytotoxicity and receptor tyrosine kinase (RET) expression in cells exposed to BP-3. The results showed that maternal BP-3 exposure was associated with offsprings HSCR in the population as well as inhibited migration of 293T and SH-SY5Y cells. Whats more, we discovered dose-response relationship between RET expression and BP-3 exposure dose, and miR-218 and some other genes involved in SLIT2/ROBO1-miR-218-RET/PLAG1 pathway were also related to BP-3 exposure. Therefore, we deduced that BP-3 influenced cell migration via SLIT2/ROBO1-miR-218-RET/PLAG1 pathway. Our study firstly revealed the relationship between maternal BP-3 exposure and HSCR as well as its potential mechanism.

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.