Aberrant expression of LncRNA-MIR31HG regulates cell migration and proliferation by affecting miR-31 and miR-31* in Hirschsprung's disease.

Hirschsprung's disease (HSCR) is a birth defect that causes a failure of the enteric nervous system to cover the distal gut during early embryonic development. Evidence shows that long non-coding RNAs (lncRNA) play important roles in HSCR. The MIR31 host gene (MIR31HG), also known as Loc554202, is a long non-coding RNA (lncRNA), which acts as the host gene of (microRNA) miR-31 and miR-31*. There have been no studies regarding its function in early developmental defects during pregnancy, and its downstream genetic receptors. We report that downregulation of MIR31HG inhibited migration and proliferation in 293T and SH-SY5Y cell lines, by suppressing miR-31 and miR-31*. Moreover, the downregulation of miR-31 and miR-31* enhanced inter-α-trypsin inhibitor heavy chain 5 (ITIH5) and the phosphatidylinositol-4, 5-bisphosphate 3-kinase catalytic gamma subunit (PIK3CG), respectively with reductions of cell migration and proliferation in 293T and SH-SY5Y cell lines. In addition, synergistic actions were observed between miR-31 and miR-31* in cell migration and proliferation. Our results demonstrated that the MIR31HG-miR-31/31*-ITIH5/PIK3CG pathway plays a role in the pathogenesis of HSCR.

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

[This corrects the article DOI: 10.7150/ijms.18392.].

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.

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. MATERIALS AND METHODS: In all eight family members of a non-consanguineous Chinese family, we performed whole-exome sequencing. Subsequently, exome sequencing of MYH14 in 72 unrelated probands with ARMs was performed. The accurate distribution of non-muscle myosin II heavy chain (NMHC II) was investigated by immunohistochemistry in serial sagittal sections of E11.5-13.5 mouse cloacal regions. RESULTS: A homozygous mutation in MYH14 was identified in the two siblings of family 1. Compound heterozygous MYH14 changes were identified in an unrelated individual. Immunohistochemical analysis suggest stronger NMHC IIC localization in the epithelium of the murine embryonic cloaca, urorectal septum and hindgut compared with another two NMHC II isoforms. CONCLUSION: This is the first identification of mutations in MYH14 as a cause of ARMs. The stronger localization of NMHC IIC in E11.5-13.5 mouse cloacal regions further supports the role of MYH14 in anorectal development.

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

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.

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.

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

BackgroundHirschsprung's 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.MethodsWe 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.ResultsMir-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.ConclusionsOur study shows the potential function of mir-939 through regulating LRSAM1 in HSCR and infers that autophagy may also confer the risk of HSCR.

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.

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.

X chromosome-wide identification of SNVs in microRNA genes and non-obstructive azoospermia risk in Han Chinese population.

Human X chromosome has higher densities of microRNAs (miRNAs) compared to the average densities on autosomes. Given that numbers of X-linked miRNAs can escape from meiotic sex chromosome inactivation (MSCI) silencing, it is proposed that X-linked miRNAs may play critical roles in the process of spermatogenesis. To test the hypothesis, we performed DNA capture sequencing of human X-linked miRNAs, which was followed by a two-stage case-control study to identify the non-obstructive azoospermia (NOA) related single nucleotide variants (SNVs) in 1107 NOA cases and 1191 fertile healthy controls. Eventually, we found rs5951785, located near hsa-miRNA-506/507, increased the risk of NOA, while rs1447393, near hsa-miRNA-510, decreased the risk of NOA. Functional analysis revealed that rs5951785 significantly inhibited cell proliferation and induced cell apoptosis. Taken together, our results demonstrated that X-linked miRNAs played important roles in the pathogenesis of NOA.