| Background:Hirschsprung’s disease(HSCR)is a congenital disease of the digestive tract caused by defects in the function of the intestinal nervous system during the embryonic period.The abnormal migration function of neural crest cells plays an important role in the pathogenesis of this disease.The enteric nervous system(ENS)is the intrinsic nervous system of the intestine,which finely regulates the intestine.When ENS is absent,there is no neurally mediated propulsion pattern and the intestine remains contracted,resulting in functional obstruction.Symptoms of congenital megacolon include constipation,vomiting,bloating,and growth disorders.Untreated diseases usually lead to childhood deaths.The current treatment is surgical removal of the intestines to remove or bypass areas lacking ENS,but many children still have problems after surgery.Although the anatomy of the Hirschsprung’s disease is simple,many clinical features remain mysterious,and diagnosis and treatment remain challenging.Studies have shown that long-chain non-coding RNA(IncRNA)plays an important role in many cellular processes,such as migration,proliferation,cell cycle,and tumor metastasis.For MIR31HG,as a long non-coding RNA,studies are mostly focused on the development and progression of cancer,and its role in embryonic development is still rare.By reviewing the MIR31HG sequence,it was found that its intron region encodes miRNA(miR)-31,and miR-31 has also been shown to be involved in tumor development,but its function in congenital gastrointestinal diseases is still unclear.Bioinformatics prediction revealed that miR-31 potentially regulates the downstream target gene ITIH5,but there are few reports on ITIH5,especially in the development of the enteric nervous system.The aim of this study was to investigate the regulatory mechanism of miR-31 mediated by MIR31HG on downstream ITIH5 and to explore its possible role in the development of Hirschsprung’s disease.Methods:We clarified mRNA and protein levels of MIR31HG,miR-31 in 90 HSCR patients and 90 controls by quantitative real-time PCR and western blot.By siRNA and miRNA inhibitor interference cell assays,the regulation of miR-31 was verified by inhibiting the expression of MIR3 IHG,and cell migration and proliferation assays were performed to detect the effects of abnormal expression of MIR3IHG and miR-31 on cell function.Based on comprehensive bioinformatics prediction and miRNA expression profiling chip results,we predict the gene related to the downstream of miR-31,and detect their expression levels in human tissues by qRT-PCR and Western blot.The binding relationship between miR-31 and ITIH5 was determined.The regulation of miR-31 on ITIH5 was verified in 293T and SH-SY5Y cell lines,and the function of high expression of ITIH5 was verified by miR-31 inhibitor+siRNA-ITIH5 co-transfection torsion test.Finally,cell function test(migration,proliferation)were performed to investigate the effect of MIR31HG on the function of nerve cells in the signaling pathway,and to analyze the possible role of MIR3IHG-miR-31-ITIH5 pathway in the development of HSCR.Results:Here we report the expression levels of MER.3lHG and miR-31 in the colonic samples of HSCR population were significantly lower than those in the control group,while the expression level of ITIH5 was higher than that in the control group.In the 293T and SH-SY5Y cell lines,the low expression of MIR31HG can promote the expression of ITIH5 and inhibit the migration and proliferation of cells by inhibiting the mRNA level of miR-31.Report gene experiments determined that ITIH5 is a target gene for miR-31.Conclusions:Our study demonstrates that down-expression of MIR31HG can inhibit the expression of miR-31,and then promote the expression of ITIH5,which affects the migration and proliferation of neural cells.The MIR31HG-miR-31-ITIH5 pathway may be associated with the occurrence of Hirschsprung’s disease. |
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