The Study On Mechanism Of MiR-204-3p And SP1 In The Proliferation And Migration Of Rat Enteric Neural Crest Stem Cells

Objective:Enteric nervous system(ENS),recently called "second brain",is an aggregation of neurons and glial cells originating from Enteric neural crest stem cells(ENCSCs),forming a relatively independent neural network in the gastrointestinal tract.Abnormal development of ENS may lead to some of the most common disorders associated with digestive tract malformation in childhood,as well as gastrointestinal motility disorders and functional gastrointestinal disorders in adulthood.In terms of the quantity and quality of intestinal neurons,the diseases caused by dysplasia of ENS can be divided into three types:intestinal neuron deletion(Hirschsprung),intestinal neuronal dysplasia and reduced number of intestinal ganglion cells.Among them,Hirschsprung(HD)is the most common disease.HD is a common congenital bowel movement disorder,mainly characterized by intestinal wall spasm caused by the absence of ganglion cells in the distal colon,which obstructs the passage of feces in the proximal colon and leads to distal intestinal wall dilation.The main symptom is constipation,and the incidence is about 1/5000.At present,as the only effective treatment for this disease,surgical resection of the distal intestinal ganglion of HD with segmental absence can reduce constipation symptoms,but there are many complications,which will affect the quality of life of the children in the future.Therefore,it is of great significance to further explore the pathogenesis of HD and to find better treatment methods based on this.It has been reported that dysplasia of ENS is caused by impaired migration of ENSCs during fetal development,which is the main cause of the absence of diseased intestinal ganglia in HD patients.It is widely believed that the neural crest stem cell migration theory accurately describes the whole process of ENS development:the proliferation and migration of ENCSCs along the intestinal tract;Differentiation of neurons and glial cells after colonization of ENCSCs;Forming ganglion in the intestinal wall;Neuronal axon and synaptic formation.Neural crest stem cells(NCSCs)are known to separate from dorsal neural crest folds during embryonic development and begin a directional migration journey to mesodermal mesenchyma,where they are finally colonized.Some of these NCSCs are specifically differentiated into neurons and glial cells of ENS,which are called ENCSCs.ENCSCs migrate,colonize,differentiate and eventually develop into ENS.The molecular regulatory mechanisms involved in these physiological processes are very complex,and the dynamic source of NCSCs migration is not very clear.Chemotaxis and epithelia-mesenchymal transition(EMT)are important mechanisms of epithelial cell migration and proliferation.In the early stage,the immunofluorescence staining analysis on the sections of rat embryos confirmed that CXCR4 was expressed on the surface of ENCSCs,and CXCR4 induced NCSCs to migrate from the neural crest to the intestinal tract along the concentration gradient of SDF-1,suggesting that SDF-1/CXCR4 axis played a regulatory role in the migration of NCSCs.Meanwhile,in NCSCs extracted from rat embryos,our team confirmed that the expression of E-cadherin was decreased and the expression of N-cadherin was increased during the development of NCSCs,implying that EMT plays a vital role in the migration of NCSCs.However,the specific regulatory mechanisms of SDF-1/CXCR4 axis and EMT-mediated biological activities such as cell adhesion,migration and proliferation are still not fully understood,and we speculate that there is a regulatory mechanism of interaction between them.MicroRNAs(miRNAs)are short endogenous RNA with a length of about 22nt discovered in recent years.MiRNA plays a molecular biological role by complementary binding with the 3'-UTR of the target mRNA,leading to regulation of the translation and expression of target genes at the post-transcriptional level.It plays an important regulatory role in ontogeny,cell differentiation,apoptosis and other physiological activities,and is related to the occurrence and development of a variety of diseases.Currently,several miRNAs have been found to be abnormally expressed in the disaffected intestinal segments of HD patients,and miRNAs play a regulatory role in the proliferation,migration,differentiation and survival of ENCSCs through different mechanisms.However,many miRNAs remain to be discovered.Therefore,it is necessary to conduct in-depth studies on the mechanism of miRNA's involvement in the developmental regulation of ENS,in order to find the miRNA molecules that play an important role,and on this basis to explore new therapeutic targets.In this study,we compared the miRNA expression profiles in the pathological and normal intestinal tissues of HD patients,and combined with bioinformatics analysis,looked for differentially expressed miRNAs.Then,the primary cells of ENCSCs were extracted from fetal rats at the peak of CXCR4 expression(i.e.,gestational age of 12 days).Key miRNAs were overexpressed in ENCSCs through adenovirus transfection,and the miRNAs were studied at the cellular and molecular levels,in order to obtain the miRNAs involved in the SDF-1/CXCR4 axis and the EMT process,and to explore the mechanism of their specific regulation of the proliferation,apoptosis and migration of ENCSCs.Methods:1.High-throughput sequencing was used to screen miRNAs differentially expressed in the pathological and normal intestinal tissues of HD patients.Collect surgical removal of the intestinal tissue from 3 children with HD,spastic intestinal tissues were aganglionic ones by immunohistochemical detection in department of pathology and were divided into lesion group,dilate intestinal tissues were ganglionic ones and were divided into normal group.The differentially expressed miRNAs were screened by high-throughput sequencing technology,and the GO and KEGG function enrichment analysis of the miRNAs with large differential multiple was performed to screen out the miRNAs that may be involved in both chemotaxis and EMT processes.Important miRNAs were verified by RT-qPCR.Target gene prediction of important miRNAs was performed using bioinformatics database.2.Extraction,culture and identification of ENCSCs.Wistar rats aged 7?9 weeks and weighing 230?280g were selected and provided by the Experimental Animal Feeding Center of XX XX Affiliated to XXX.The rats were raised in an SPF animal feeding room(room temperature 22±2?;Humidity 55+/-5?;Maintain a 12-hour day/night cycle).According to the ratio of male and female 4:1,the cages were combined at midnight,and vaginal smear was performed between 8:00 to 9:00 the next day.The woman who saw a large number of spermatozoa under light microscope was recorded as the gestation day 0(Embryonic 0,E0).On the 12th day of gestation(E12),the fetus was dissected,and the embryonic intestinal tissues were removed with microscopic instruments under the microscope.After physical fragmentation and chemical enzyme digestion,the cells were resuspended in the special culture medium for neural stem cells and inoculated into the culture flask.The cells were cultured in an incubator at 37? and 5%CO2 saturated humidity.After primary incubation for 24h,the floating dead cells were discarded,the liquid was replaced with 2/3 of the amount,and the culture was continued.The neurosphere formation of the cells was observed with an inverted microscope.The primary cells were identified by morphological and immunological methods(immunofluorescence and flow cytometry).3.Effects of miR-383-3p,miR-490-5p,miR-200c-3p and miR-204-3p on the expression levels of CXCR4,ZEB2 and E-cadherin.ENCSCs were transfected with adenovirus in rats.After the overexpression of miR-383-3p,miR-490-5p,miR-200c-3p and miR-204-3p,respectively,RT-qPCR was used to detect the overexpression efficiency and determine the optimal transfection concentration,and the expression levels of CXCR4,ZEB2 and E-cadherin were detected.4.The effect of miR-204-3p on SP1 expression.After the overexpression of miR-204-3p in ENCSCs of rats,SP1 expression was detected by RT-qPCR.5.Effects of miR-204-3p and SP1 on the function of rat ENCSCs cells.Three siRNAs were designed for SP1 and packaged with adenovirus,and the interference efficiency was detected by RT-qPCR after transfection with ENCSCs,and the siRNAs with the best interference effect were selected for subsequent experiments.After overexpression of miR-204-3p or SP1 knockdown,the changes of cell proliferation,cell flow apoptosis and cell migration were detected,respectively.6.Effects of SP1 on the expression of CXCR4,E-cadherin and miR-204-3p.After SP1 interference,the expression levels of CXCR4,E-cadherin and miR-204-3p were detected by RT-qPCR.7.Effect of CXCR4 on the function of ENCSCs cells in rats.Three siRNAs were designed for CXCR4 and packaged with adenovirus.The interference efficiency was detected by RT-qPCR after transfection with ENCSCs,and the siRNAs with the best interference effect were selected for subsequent experiments.After CXCR4 was knocked down,cell proliferation,flow cell apoptosis and cell migration ability were detected.8.Statistical analysis.Experimental data were expressed as mean±standard deviation and statistically analyzed using SPSS 21.0 and Prism 7.0 software.For comparison between two groups,Student's t test was used;One-way ANOVA analysis and Tukey post-hoc analysis were used for comparison between groups.p<0.05 is considered statistically significant.Results:1.High-throughput sequencing results showed that 277 significantly upregulated miRNAs and 254 significantly down-regulated miRNAs were found in both pathological and normal intestinal tissues of HD children.GO analysis and KEGG analysis were performed on the 30 miRNAs with the highest differential ratio,and it was found that miR-383-3p,miR-490-5p,miR-200c-3p and miR-204-3p were related to both EMT and cell chemotaxis.The expression trend of these 4 miRNAs was verified by RT-qPCR,which was consistent with the basic results of sequencing.Through the prediction of target genes,it was found that miR-204-3p had the most extensive association with target genes.2.After the overexpression of miR-200c-3p and miR-204-3p in ENCSCs,respectively,the expression of CXCR4 and ZEB decreased,while the expression of E-cadherin increased;However,after the overexpression of miR-383-3p and miR-490-5p,the expression of CXCR4 was not significantly changed,the expression of ZEB2 was decreased and the expression of E-cadherin was increased.3.After the overexpression of miR-204-3p in ENCSCs of rats,the expression of SP1 decreased.4.Overexpression of miR-204-3p in rat ENCSCs can lead to reduced proliferation,increased apoptosis and reduced migration of rat ENCSCs.Knocking down SP1 produced a similar result.5.After SP1 knockdown in ENCSCs of rats,the expression of CXCR4 decreased and the expression of E-cadherin increased,and after SP1 knockdown,it was found that the expression of miR-204-3p decreased.6.Knocking down CXCR4 in rat ENCSCs can cause reduced proliferation ability,increased apoptosis,and reduced migration ability of rat ENCSCs.Conclusion:1.There are many differentially expressed miRNAs in the pathological and normal intestinal tissues of children with congenital megacolon,which may be related to the occurrence and development of children with congenital megacolon,and further study is needed.2.After overexpression of miR-204-3p or knockdown of SP1 or CXCR4,the proliferation,apoptosis and migration ability of ENCSCs in rats were reduced,suggesting that miR-204-3p,SP1 and CXCR4 are all involved in the regulation of proliferation,apoptosis and migration of ENCSCs.3.MiR-204-3p may participate in the process of SDF-1/CXCR4 axis and EMT by regulating its potential target gene SP1.