Function And Mechanism Of MiR-140-5P In Hirschsprung’s Disease

Hirschsprung’s disease(HSCR),also called Intestinal gangliocytosis,is a neural crest cell-derived disease and a polygenic genetic disease,and is the most common congenital intestinal motility disease in children.The incidence rate is about 1/4000-5000 of living newborns,the male to female ratio is about 4:1,and there is a genetic predisposition.The main cause is that during the development of embryonic nerves,the enteric neural crest cells(ENCCs)are impaired in migration,and the development of the enteric nervous system stagnates,leading to the intramural and submucosal ganglia of the intestine with different length,the affected intestinal segment is abnormally contracted and narrowed,and the proximal colon is compensated for expansion and hypertrophy,forming a giant colon.Hirschsprung’s disease is mainly manifested by symptoms of low intestinal obstruction such as refractory constipation and bloating,which is a congenital disease that seriously affects the health of children.At present,the etiology and pathogenesis of the congenital megacolon are still unclear.Surgery is still the only method to cure the megacolon.However,the operation has a greater trauma to the child,more postoperative complications,and some complications are more difficult to handle and serious which can affect children’s quality of life in the future.Therefore,in addition to surgical treatment,whether to find other treatment methods for radical Hirschsprung’s disease is the focus of many scholars’ research in recent years.According to current research and findings,HSCR is caused by a combination of factors,and genetic factors are the most important.In recent years,the genetic research of HSCR has made significant progress.A large number of research results show that there are abnormal expressions of multiple genes and signaling pathways in HSCR patients.So far,molecular genetic analysis has identified some relevant genes in the occurrence and development of HSCR,such as:proto-oncogene RET,glial cell line-derived neurotrophic factor(GDNF),endothelin B receptor(EDNRB),endothelin 3(EDN3),endothelin converting enzyme 1,ECE1,neurotrophin(NTN),SOX10,Phox2B,KIAA1279,SIP1(smad2 interacting proteinl),NRG1,GFRα1,NRTN,TCF4,PSPN,ZFHX1B,L1CAM,SEMA3C,SEMA3D,etc.The above genes all play an important role in the differentiation and migration of neural crest stem cells,and gene defects can significantly interfere with the migration and differentiation of neural kurtocytes.If the colonization of intestinal neural crest cells in the intestine,that is,the process of nerve cell proliferation,migration,and differentiation,is impaired,then Hirschsprung’s disease will form.MicroRNAs(miRNA)is a non-coding short-sequence single-stranded RNA molecule composed of 21-25 nucleotides,which is an endogenous small molecule RNA.MicroRNAs can specifically bind to the 3 ’ non-coding region(3’UTR)of its target gene mRNA molecule in the form of base pairing,which in turn triggers mRNA degradation or translation inhibition,and finally exerts gene regulation at the post-transcriptional level.Current research finds that miRNA plays an important role in many life processes,including cell colonization,apoptosis,organ development,stress response,and the formation of various diseases.miRNAs are essential for the development of neurons and the maintenance of normal functions.Therefore,miRNAs may also be closely related to the development of the enteric nervous system,and there have been reports that some miRNAs are involved in the differentiation,proliferation,migration and regulation of enteric neural crest cells,the whole process of death is related to the formation of Hirschsprung’s disease in children.At present,there are few studies on miRNAs in Hirschsprung’s disease.Therefore,an in-depth discussion of the role and regulatory mechanism of miRNAs will help further clarify the pathogenesis and provide new ideas for its targeted therapy.We used miRNA gene chip technology to detect differentially expressed miRNAs in the intestinal tract tissues of the megacolon stenosis and dilated segments,and initially established miRNA expression profiles of megacolon tissue.The criterion for significant difference in miRNA expression was Fold Change>2 and p<0.05.It was found that compared with the megacolon dilated intestine,there were 24 miRNAs up-regulated and 16 down-regulated in the narrowed bowel.MiR-140-5p,which has obvious differential expression in down-regulated miRNA,has not been reported,and has a close relationship with neural development,was selected as the research object.Selected SH-SY5Y(human neuroblastoma cells)and 293T(human kidney epithelial cells)cell lines.Lentivirus interfered with miR-140-5p expression in SH-SY5Y and 293T cells.In vitro cytology and proteomics tests to verify the effects of miR-140-5p on the proliferation,migration and differentiation of intestinal neural crest cells,and to explore the role of miR-140-5p in the formation of Hirschsprung’s disease providing a further theoretical basis for targeted therapy.Materials and MethodCollected surgical specimens of intestinal stenosis and dilated segments of 32 children with Hirschsprung’s disease who were treated and operated in Shandong Provincial Hospital,including 22 males and 10 females;aged 3 to 5 years.Preoperative barium enema,anorectal manometry,and postoperative pathological confirmation.Five cases were used for chip detection and 27 cases were used for extended sample size verification.The chip was provided by Shanghai Kangcheng Biotechnology Co.,Ltd.1.Screening using miRNAs gene chips(chips provided by Shanghai Kangcheng Biological Technology Co.,Ltd.),and discovering differentially expressed miRNAs.The significant difference in miRNAs expression is judged by Fold Change>2 and p<0.05.2.By screening and analyzing the results of miRNA gene chips,we finally selected miR-140-5p,which has not been reported yet and has a close relationship with neural development,as the research object,and further verified by qRT-PCR.3.Select SH-SY5Y cells(human neuroblastoma cells)and 293T cells(human renal epithelial cells)for in vitro cytology experiments.Lentiviral interference down-regulated the expression of miR-140-5p in SH-SY5Y and 293T cells,and divided them into blank control group,virus-free transfection group,and miR-140-5p knockout group.qRT-PCR to detect the efficiency of cell transfection;CCK-8 test to detect the proliferation of cells in each group after transfection;Transwell cell migration test and scratch test to detect the changes in cell migration capacity of miR-140-5p Impact;Western blotting detection of miR-140-5p knockout and autophagy-related proteins(Bax,Beclin-2,cleared-caspase-3,pro-caspase-3,cleared-parp-1)expression;Flow cytometry was used to detect the changes of apoptosis in each group;4.Using bioinformatics software to predict the target gene of miR-140-5p is EGR2;At the same time,double luciferase experiments were performed to verify the regulatory mechanism of miR-140-5p on its target gene EGR25.Western blotting and immunohistochemistry to detect the relative expression of EGR2 in Hirschsprung disease and normal intestinal tissue;Western blotting to detect the expression of EGR2 in each group of cells;6.Western blotting to detect the expression of AKT signaling pathway related proteins(p-AKT,AKT),and relevant experiments were conducted to analyze the role of AKT signaling pathway in the pathogenesis of HSCR.7.Statistical method:SPSS 19.0(SPSS Company,Chicago,United States)statistical software is used.The experimental data of this subject were statistically analyzed using Student’s t-test,χ2 test,or Fisher’s exact method.P<0.05 was considered statistically significant.Results1.Results of miRNA gene chip detection in children with Hirschsprung’s diseaseThe results of gene chip testing showed that compared with the expanded tissue,there were 40 differentially expressed miRNAs in the narrow tissue(the significant difference criterion was Fold Change>2 and p<0.05.),Of which 24 miRNAs were highly expressed,such as:MiRNA-483-5P,miRNA-218-1,miRNA-195,miRNA-124,miRNA-25,miRNA-133a,etc.The specific results are shown in Table 1-1;16 types of miRNAs are low expressed,such as:miRNA-141-3P,miRNA-200a,miRNA-206,miRNA-140-5P,etc.,the differences are statistically significant(P<0.05),the specific results are shown in Table 1-2.MiR-140-5p,which has obvious differential expression in down-regulated miRNAs,has not been reported and has a close relationship with neurodevelopment,is selected as the research object.2.Verification of in vitro cytology experiments and related functional tests(1).CCK8 test showed that the down-regulation of miR-140-5p reduced the proliferation of cells;Western blotting detection of apoptosis-related proteins showed that down-regulation of miR-140-5p promoted apoptosis;flow cytometry results showed that miR-140-5p Down-regulation promoted the apoptosis of SH-SY5Y and 293T;scratch experiments showed that the down-regulation of miR-140-5p led to a decrease in cell migration ability;and the Transwell experiment results further showed that the down-regulation of miR-140-5p reduced the cell migration ability.(2).Use the target gene prediction software Targetscans to predict that the target gene of miR-140-5p acting on the megacolon may be EGR2.The dual luciferase experiment proved that miR-140-5p can exert an inhibitory effect on the 3’UTR of EGR2.Co-transfection of sh-sy5y cells with EGR2 siRNA plasmid and silencing mir-140-5p lentivirus further demonstrated that mir-140-5p can participate in the pathogenesis of congenital megacolon by regulating EGR2.(3).Western blotting and immunohistochemistry results showed that the expression level of EGR2 in the narrow bowel tissue was significantly up-regulated,and the expression of miR-140-5p was significantly negatively correlated with EGR2;The expression of EGR2 increased significantly after miR-140-5p was knocked down in SH-SY5Y and 293T,and the difference was statistically significant(P<0.05).(4).Western blotting results showed that SH-SY5Y and 293T were down-regulated after the knockout of miR-140-5p,signaling pathway-related protein P-AKT,suggesting that the down-regulation of miR-140-5p may inhibit the AKT signaling pathway through its target gene EGR2,and thus inhibit The proliferation and migration ability of SH-SY5Y and 293T also promote their apoptosi s.After the use of Akt signaling activator,cell apoptosis was inhibited and cell proliferation and migration capacity increased,indicating that mir-140-5p can participate in the pathogenesis of congenital megacolon through the Akt signaling pathwayConclusion1.MiRNA expression differences exist in the narrow and dilate tracts of the megacolon.A variety of miRNAs are involved in the occurrence and development of Hirschsprung’s disease,which needs further study.2.miR-140-5p is down-regulated in children with congenital megacolon stenosis and inhibits the proliferation,migration and differentiation of intestinal neural crest cells in the intestine,thereby playing a role in the occurrence and development of HSCR.3.miR-140-5p may inhibit the AKT signaling pathway through its target gene EGR2 and participate in the pathogenesis of HSCR.