A Preliminary Study On The Pathogenesis Of MicroRNA-517a In Hirschsprung’s Disease

Background:Hirschsprung’s disease(HSCR)is a congenital gastrointestinal disease caused by the abnormal function of the enteric nervous system in the early stage of embryonic development.In the pathogenesis,abnormal migration of neural crest cells plays an important role in the pathogenesis of HSCR.The clinical manifestations of Hirschsprung’s disease have multiple symptoms,including long-term constipation,intermittent vomiting,repeated abdominal distension and growth retardation.Untreated diseases usually affect the growth and development of children,and even die.The current treatment method is mainly surgery,which removes the diseased intestinal tube and rebuilds the integrity and function of the intestine.However,there are still some children who suffer from feces and Complications such as constipation.In recent years,with the improvement of the medical level and the progress of related molecular biology research,the diagnosis and treatment of HSCR has made great progress,but there are still many problems about the pathogenesis to be solved.Studies have shown that microRNA(miRNA)plays an important role in many physiological or pathological processes,such as migration,proliferation,cycle,apoptosis,and autophagy.Combined with the existing miRNA chip results of the research group,we found that miR-517a is abnormally expressed in HSCR.MiR-517a has been found to be involved in the occurrence and development of multiple diseases,including cancer,and affect cell migration,but research on its role in embryonic development is still rare.Through bioinformatics prediction,it was discovered that miR-517a potentially regulates downstream target gene PTEN(gene of phosphate and tension homology deleted on chromsome ten,PTEN).PTEN is one of the tumor suppressor genes.Most studies suggest that it is involved in the occurrence of cancer,but there are few reports on its regulatory mechanism during embryonic development,especially The role in the development of the enteric nervous system during the embryonic stage has not been reported yet.This experiment aims to study the regulatory mechanism of miR-517a on downstream PTEN,and to explore the possible role of this mechanism in the occurrence of Hirschsprung’s disease.METHODS:36 cases of HSCR stenosis intestines and 34 cases of gender and age-matched normal control colon tissue samples,RNA was extracted and reverse transcribed into cDNA.The colon tissues of the case group and the control group were determined by qRT-PCR(quantitative real-time PCR)The mRNA expression level of miR-517a.In the in vitro cell experiment,the SH-SY5Y cell line was used,after the interference of miRNA inhibitor,the influence of the abnormal expression of miR-517a on cell function was detected through cell migration and proliferation experiments.Take miR-517a as the upstream,integrate the results of bioinformatics prediction and protein expression profile chip,predict the downstream related genes of miR-517a,use qRT-PCR and Western blot to detect its expression level in human tissues,and pass the reporter gene experiment Determine the binding relationship between miR-517a and PTEN.The regulation of miR-517a on PTEN was verified in SH-SY5Y cell line,and the effect of high-expression PTEN on cells was verified by miR-517a inhibitor+siRNA-PTEN co-transfection torsion experiment.Cell function experiments(migration,proliferation,cell cycle,apoptosis)were carried out to explore the effect of miR-517a,PTEN in the signal pathway on nerve cell function.Finally,we analyze the possible role of miR-517a/PTEN pathway in the occurrence of HSCR by RNA-seq.RESULTS:The expression level of miR-517a in the colon segment of HSCR population was significantly lower than that in the control group,while the expression level of PTEN was higher than that in the control group.In the SH-SY5Y cell line,low expression of miR-517a promotes PTEN expression and affects cell migration and proliferation.The highly expressed miR-517a inhibited the expression of PTEN and promoted cell migration and proliferation.Based on RNA-seq data,we found that miR-517a might be involved in the regulation of growth and development,stem cell pluripotency,and nucleic acid binding transcription factor activity,suggesting that it might be involved in the regulation of early embryonic development.Conclusion:Low expression of miR-517a can promote the expression of PTEN,which affects the migration and proliferation of neural cells.The miR-517a-PTEN pathway may be associated with the occurrence of Hirschsprung’s disease,and miR-517a may be closely related to embryonic development...