Chunxia Du

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.

Microarray expression profiling of dysregulated long non-coding RNAs in Hirschsprung's disease reveals their potential role in molecular diagnosis

Hirschsprungs disease (HSCR) is one of the common digestive disorders in the new born. Long non‐coding RNAs (lncRNAs) play an important role in various biological processes. However, knowledge on lncRNAs in HSCR is limited.

Apoptotic neuron-secreted HN12 inhibits cell apoptosis in Hirschsprung's disease

Perturbation in apoptosis can lead to Hirschsprung’s disease (HSCR), which is a genetic disorder of neural crest development. It is believed that long noncoding RNAs (lncRNAs) play a role in the progression of HSCR. This study shows that apoptotic neurons can suppress apoptosis of nonapoptotic cells by secreting exosomes that contain high levels of HN12 lncRNA. Elevated exogenous HN12 in nonapoptotic cells effectively inhibited cell apoptosis by maintaining the function of mitochondria, including the production of ATP and the release of cytochrome C. These results demonstrate that secreted lncRNAs may serve as signaling molecules mediating intercellular communication in HSCR. In addition, high HN12 levels in the circulation worked as a biomarker for predicting HSCR, providing a potential, novel, noninvasive diagnostic approach for early screening of HSCR.

Downregulated Expression of Long Non-Coding RNA LOC101926975 Impairs both Cell Proliferation and Cell Cycle and Its Clinical Implication in Hirschsprung Disease Patients.

Background: Long non-coding RNAs (lncRNAs) have been reported to participate in various diseases. Hirschsprung disease (HSCR) is a common digestive disease in the new born. However, the relationship between lncRNAs and HSCR remains unclarified. Methods: We used qRT-PCR to detect the relative expression of LOC101926975 in 80 pairs of HSCR bowel tissues and matched normal bowel tissues. CCK-8 assay, transwell assay and flow cytometry were then used to evaluate the function in vitro by knocking down the LOC101926975 in SK-N-BE(2) cells. Receiver operating characteristic (ROC) curve was used to evaluate the potential diagnostic value of LOC101926975. Results: LOC101926975 was significantly downregulated in HSCR tissues with excellent correlation with FGF1. Dysregulation of LOC101926975 suppressed cell proliferation and induced G0/G1 arrest without impact on cell apoptosis or migration. Meanwhile, the AUC of LOC101926975 was 0.900 which presented great diagnostic value. Conclusions: Our study firstly investigates the potential function of LOC101926975 in HSCR and infers that LOC101926975 can distinguish HSCR from the normal ones.

Downregulation of lncRNA MEG3 and miR-770-5p inhibit cell migration and proliferation in Hirschsprung’s disease

The long noncoding RNA (lncRNA) MEG3 is involved in various biological processes including cell migration and cell proliferation. In present study, it was found that MEG3 and the intronic miR-770-5p were decreased in samples from HSCR patients. Besides, knockdown of MEG3 and miR-770-5p suppressed cell migration and proliferation, while cell cycle and apoptosis were not affected in human 293T and SH-SY5Y cells. SRGAP1 mRNA and protein upregulation was inversely correlated with miR-770-5p expression in tissue samples and cell lines, which was confirmed to be a target gene of miR-770-5p by dual-luciferase reporter assay. Moreover, silencing of SRGAP1 rescued the inhibition of cell migration and proliferation induced by MEG3 siRNA and miR-770-5p inhibition. The present study elucidates a novel mechanism of the development of HSCR and shows that the MEG3/miR-770-5p/SRGAP1 pathway plays a vital role in the pathogenesis of HSCR.The long noncoding RNA (lncRNA) MEG3 is involved in various biological processes including cell migration and cell proliferation. In present study, it was found that MEG3 and the intronic miR-770-5p were decreased in samples from HSCR patients. Besides, knockdown of MEG3 and miR-770-5p suppressed cell migration and proliferation, while cell cycle and apoptosis were not affected in human 293T and SH-SY5Y cells. SRGAP1 mRNA and protein upregulation was inversely correlated with miR-770-5p expression in tissue samples and cell lines, which was confirmed to be a target gene of miR-770-5p by dual-luciferase reporter assay. Moreover, silencing of SRGAP1 rescued the inhibition of cell migration and proliferation induced by MEG3 siRNA and miR-770-5p inhibition. The present study elucidates a novel mechanism of the development of HSCR and shows that the MEG3/miR-770-5p/SRGAP1 pathway plays a vital role in the pathogenesis of HSCR.

Mutations of MYH14 are associated to anorectal malformations with recto-perineal fistulas in a small subset of Chinese population

Background: Anorectal malformations (ARMs) are among the most commonly congenital abnormalities of distal hindgut development, ranging from anal stenosis to anal atresia with or without fistulas and persistent cloaca. The etiology remains elusive for most ARM cases and the majority of genetic studies on ARMs were based on a candidate gene approach.

Negative feedback circuitry between MIR143HG and RBM24 in Hirschsprung disease.

Hirschsprung disease (HSCR) is a genetic disorder of neural crest development. It is also believed that epigenetic changes plays a role in the progression of this disease. Here we show that the MIR143 host gene (MIR143HG), the precursor of miR-143 and miR-145, decreased cell proliferation and migration and forms a negative feedback loop with RBM24 in HSCR. As RBM24 mRNA is a target of miR-143, upregulation of RBM24 upon an increase in the level of MIR143HG could be attributed to sequestration of miR-143 by MIR143HG (sponge effect). The RBM24 protein was shown to bind to MIR143HG, and subsequently, accelerated its degradation by destabilizing its transcript and facilitating its interaction with Ago2, thus forming a negative feedback between MIR143HG and RBM24. In addition, experiments using siRNA against DROSHA indicated that RBM24 could promote the biogenesis of miR-143. This feedback loop we describe here represents a novel mode of autoregulation, with implications in HSCR pathogenesis.

LncRNA AFAP1-AS Functions as a Competing Endogenous RNA to Regulate RAP1B Expression by sponging miR-181a in the HSCR: Erratum

Background: Long noncoding RNAs (lncRNAs) have recently emerged as important regulators in a broad spectrum of cellular processes including development and disease. Despite the known engagement of the AFAP1-AS in several human diseases, its biological function in Hirschsprung disease (HSCR) remains elusive. Methods: We used qRT-PCR to detect the relative expression of AFAP1-AS in 64 HSCR bowel tissues and matched normal intestinal tissues. The effects of AFAP1-AS on cell proliferation, migration, cell cycle, apoptosis and cytoskeletal organization were evaluated using CCK-8, transwell assay, flow cytometer analysis and immunofluorescence, in 293T and SH-SY5Y cell lines, respectively. Moreover, the competing endogenous RNA (ceRNA) activity of AFAP1-AS on miR-181a was investigated via luciferase reporter assay and immunoblot analysis. Results: Aberrant inhibition of AFAP1-AS was observed in HSCR tissues. Knockdown of AFAP1-AS in 293T and SH-SY5Y cells suppressed cell proliferation, migration, and induced the loss of cell stress filament integrity, possibly due to AFAP1-AS sequestering miR-181a in HSCR cells. Furthermore, AFAP1-AS could down-regulate RAP1B via its competing endogenous RNA (ceRNA) activity on miR-181a. Conclusions: These findings suggest that aberrant expression of lncRNA AFAP1-AS, a ceRNA of miR-181a, may involve in the onset and progression of HSCR by augmenting the miR-181a target gene, RAP1B.

MicroRNA-939 inhibits cell proliferation via targeting LRSAM1 in Hirschsprung’s disease

Background Hirschsprungs disease (HSCR) is a common digestive disease caused by impaired development of neural crest cells. Some studies have revealed the roles of microRNA (miRNA) in various diseases. But the function of miRNA in HSCR needs further investigation. Methods We adopted qRT-PCR and immunoblot analyses to explore the relative expression of miR-939 and LRSAM1 in 80 HSCR bowel tissues and 80 normal bowel tissues. CCK-8 assay, transwell assay and flow cytometry were used to evaluate the function of miR-939 by overexpression of miR-939 in 293T, SK-N-BE(2), SH-SY5Y cell lines. The direct connection between miR-939 and LRSAM1 was validated by dual-luciferase reporter assay. We also investigated the autophagy level via immunoblot analyses. Results Mir-939 was significantly upregulated in HSCR tissues with decreased expression of LRSAM1. Overexpression of miR-939 suppressed cell proliferation without affecting cell apoptosis, cell cycle or cell migration. And LRSAM1 exerted similar function. Autophagy was impaired in HSCR tissues compared with control samples. Mir-939 did not inhibit the autophagy although it decreased the expression of LRSAM1. Conclusions Our study shows the potential function of mir-939 through regulating LRSAM1 in HSCR and infers that autophagy may also confer the risk of HSCR.

Lipopolysaccharide enhances ADAR2 which drives Hirschsprung's disease by impairing miR-142-3p biogenesis

Researches over the past decade suggest that lipopolysaccharide is a dominant driver of gastrointestinal motility and could damage the enteric neuron of rat or porcine. However, it remains poorly defined whether LPS participates in Hirschsprungs disease (HSCR). Here, we discovered that LPS increased in HSCR tissues. Furthermore, LPS treatment suppressed the proliferation and differentiation of neural precursor cells (NPCs) or proliferation and migration of human 293T cells. ADAR2 (adenosine deaminase acting on RNA2)‐mediated post‐transcriptional adenosine‐to‐inosine RNA editing promotes cancer progression. We show that increased LPS activates ADAR2 and subsequently regulates the A‐to‐I RNA editing which suppresses the miR‐142 expression. RNA sequencing combined with qRT‐PCR suggested that ADAR2 restrain cell migration and proliferation via pri‐miR‐142 editing and STAU1 up‐regulation. In conclusion, the findings illustrate that LPS participates in HSCR through the LPS‐ADAR2‐miR‐142‐STAU1 axis.