ABSTRACT
Background LncRNAs are known to take part in normal brain functions and nervous system diseases. Little evidence has pointed to the dysregulation of lncRNAs in congenital intestinal atresia. We aimed to investigate the underlying molecular mechanism of congenital intestinal atresia that involves in lncRNA-MEG3. Materials and methods The expressions of LncRNA-MEG3, miR-211-5p and GDNF were determined by the qRT-PCR and Western blot assay when appropriate. The results were verified in intestinal atresia Wistar rat model and bone marrow derived stem cell (BMSCs)-derived into intestinal ganglion cells. RNA immunoprecipitation and RNA pull-down assays were performed to analyze the regulatory mechanism between MEG3 and miR-211-5p. The effects of MEG3 on the cell proliferation and apoptosis of isolated intestinal ganglion cells were detected with an MTT assay and flow cytometry, respectively. Results The expression of MEG3 was detected to be declined in congenital intestinal atresia tissues at clinic and animal levels. MEG3 promoted the differentiation of BMSCs into intestinal ganglion cells and regulated GDNF expression in retinal ganglion cells (RGC-5 cells) via targeting miR-211-5p. Hypoxia induced the apoptosis of intestinal ganglion cells via MEG3/miR-211-5p/GDNF axis. Conclusion MEG3 promoted the differentiation of BMSCs into intestinal ganglion cells and inhibited the apoptosis of intestinal ganglion cells under the exposure of hypoxia to protect against CIA injury via directly regulating miR-211-5p/GDNF axis.
Subject(s)
Glial Cell Line-Derived Neurotrophic Factor/physiology , Intestinal Atresia/metabolism , MicroRNAs/physiology , RNA, Long Noncoding/biosynthesis , Animals , Cell Differentiation/physiology , Cells, Cultured , Female , Ganglion Cysts/metabolism , Ganglion Cysts/pathology , Humans , Infant, Newborn , Intestinal Atresia/pathology , Male , Mesenchymal Stem Cells/metabolism , Mesenchymal Stem Cells/pathology , Pregnancy , Rats , Rats, Wistar , Retinal Ganglion Cells/metabolism , Retinal Ganglion Cells/pathologyABSTRACT
Necrotizing enterocolitis (NEC) is a potentially fatal illness in premature neonates. Tumor necrosis factor-α (TNF-α) and autophagy are associated with the pathogenesis of NEC. This study aimed to explore whether TNF-α might regulate apoptosis in neonatal NEC model cells IEC-6 via regulation of autophagy. NEC rat model was induced by hand feeding and exposure to asphyxia/cold-stress for histologic examination. The NEC in vitro model (IEC-6/NEC cells) was established by stimulating the intestinal epithelial cell line IEC-6 with lipopolysaccharide (LPS, 100 µg/mL) for 3 h to investigate the effects of TNF-α on IEC-6 proliferation and apoptosis. In this study, NEC rats showed decreased proliferating cell nuclear antigen (PCNA) expression, increased TUNEL-positive cells, higher expression of TNF-α, p-ERK1/2, and autophagy-related proteins in rat small intestine compared with their controls. Additionally, the LPS-stimulated IEC-6/NEC cells showed a significantly decreased proliferation and increased apoptosis compared with the control cells. Furthermore, the LPS-stimulated IEC-6/NEC cells exhibited enhanced autophagy level, as evidenced by a dose-dependent increase in Beclin-1 protein expression, LC3II/LC3I ratio and accumulation of MDC-positive autophagic vacuoles. Moreover, inhibition of autophagy by wortmannin or LY294002 significantly abolished the LPS-mediated decreased proliferation and increased apoptosis of IEC-6/NEC cells. Results also showed that inhibition of ERK1/2 pathway using U0126 significantly inhibited TNF-α-induced autophagy. Furthermore, the TNF-α-mediated inhibition of IEC-6 proliferation and promotion of IEC-6 apoptosis was abolished by U0126. Our findings demonstrated that TNF-α might induce autophagy through ERK1/2 pathway to regulate apoptosis in neonatal NEC cells IEC-6. Our study enhances our understanding of neonatal NEC pathogenesis.
Subject(s)
Apoptosis , Autophagy , Enterocolitis, Necrotizing/enzymology , Enterocolitis, Necrotizing/pathology , Extracellular Signal-Regulated MAP Kinases/metabolism , MAP Kinase Signaling System , Models, Biological , Tumor Necrosis Factor-alpha/metabolism , Animals , Animals, Newborn , Cell Line , Cell Proliferation , Intestine, Small/pathology , MAP Kinase Signaling System/drug effects , Phosphorylation , Rats, Sprague-DawleyABSTRACT
Levels of IRS-4 (insulin receptor substrate-4) in hepatoblastoma cells from the patients, HepG2 and HuH-6 cell lines were measured by Western Blot analysis, and they were all found significantly higher than those of normal hepatic cells. When the HepG2 and HuH-6 cells cultivated in vitro were treated with IRS-4 and infected with recombinant adenovirus containing IRS-4 gene, the proliferation and migration of the cells were significantly improved. Meanwhile, the expressions of PI3K (phosphatidylinositol 3'-kinase), pS473 Akt (Protein Kinase B) and pThr308 Akt were positively regulated by IRS-4 overexpression. These results indicated that IRS-4 was very likely to be involved in PI3K/Akt path way, which was further confirmed by the fact that the PI3K/Akt blocker LY294002 attenuated the proliferation and migration of the hepatoblastoma cells. In conclusion, IRS-4 enhanced the proliferation and migration of the hepatoblastoma cells via PI3K/Akt pathways.
