| Objective: Anorectal malformations(ARMs)is one of the most common congenital malformations of the digestive tract in the world,with an incidence of about1/1500～1/5000.It is usually accompanied by abnormalities of the genitourinary and cardiovascular systems.Long-term follow-up found that some patients with anorectal malformation had postoperative defecation dysfunction,including fecal incontinence and constipation,which seriously affected the long-term quality of life and physical and mental health of patients.The mechanism of ARMs is still unclear.Therefore,it is of great importance to clarify the pathogenic mechanism of ARMs as soon as possible and provide a theoretical basis for the early diagnosis and treatment of ARMs.Human ARMs refers to various intestinal malformations with different phenotypes caused by abnormal hindgut development when the embryo grows to 4-8 weeks,such as anal atresia and rectourethral fistula.Therefore,it is very difficult to obtain clinical samples at this time.It has been found that rat embryos with Ethylenethiourea(ETU)induction can have abnormal cloacal development from gestation day(GD)14 to GD16 and show ARMs phenotype very similar to human.Therefore,the ETU-induced rat embryo ARMs animal model is the most commonly used method to study anorectal malformation,laying a foundation for in-depth study of the pathogenesis of ARMs.Epidemiology and animal experiments show that genetic factors are important causes of ARMs.Among them,Wnt signaling pathway plays a crucial role in early embryonic development,organogenesis and other physiological processes,and Wnt signaling pathway is a key factor in regulating digestive tract development.For a long time,in the study of anorectal malformation,Wnt signaling pathway has been a hot topic of scholars’ research and discussion.Knockout of some key genes in Wnt signaling pathway,such as Wnt5 a gene knockout mice and β-catenin gene knockout mice,will cause pathological changes similar to human ARMs in animals.However,it remains unclear which other molecules upstream of the Wnt pathway regulate the expression of these key encoding genes.Circular RNA(circRNA)is a newly discovered endogenous non-coding RNA(nc RNA),which mainly exists in mammalian cytoplasm or exosomes.Circ RNA is a covalent closed ring structure that lacks both 5’-3’ polarity and poly-A tail.This special structure makes circRNA less susceptible to degradation by RNA exonucases,RNase R and branching enzymes,making circRNA more stable than linear transcripts.Current research most focuses on the biological function of circRNA,which can regulate the transcription and translation of coding genes by interacting with RNA polymerase II through RNA-binding proteins in place of m RNA splicing patterns.In addition,circRNA can also act as a "sponge" of miRNA to competitively inhibit the expression of coding genes.This mechanism of ce RNA forming the circRNA-miRNA-m RNA regulatory axis is the focus of current studies on the biological functions of circRNA.Recent studies have shown that circRNA plays an important regulatory role in embryonic development,apoptosis,differentiation and proliferation,and a large number of abnormally expressed circRNA are involved in the occurrence and development of gastrointestinal diseases,which can be used as a potential biomarker for diagnosis and prognosis.However,there are few studies on the role of circRNA in the genesis and development of ARMs,and the exact molecular mechanism of circRNA is still unclear.Methods: 1.Part I: High-throughput sequencing method was used to detect and verify circRNA differentially expressed in posterior gut tissues of normal and anorectal malformation rats.The experimental Animal Center of Shengjing Hospital of China Medical University provided adult female Wistar rats(Number: 36;Age: 7-8 weeks;Body weight: 230-280 g)were reared in the SPF animal laboratory of the Key Laboratory of Congenital Malformation,Ministry of Health(room temperature: 22±2℃;Humidity: 55±5%;Light/dark cycle per 12 hours;Free access to water and food).Eighteen rats were randomly selected and gavaged 125 mg/kg 1% ETU(ARM group)by single gavage on Gestational day 10(GD10),and the remaining 18 rats were given the same amount of normal saline(normal group)as control.At the critical time points of anorectal development(GD14,GD15 and GD16)of rat embryos,hysterectomy was performed to obtain fetal hindgut samples for high-throughput sequencing,and circRNA in the normal and ARM groups were fully detected.The expression trend of circRNA in the two groups was analyzed over time.Differential expression analysis and cluster observation were conducted between the two groups,and the circRNA-miRNA-m RNA regulatory network was constructed.GO and KEGG functional enrichment analysis was performed on source genes and targeted regulatory genes,so as to comprehensively explore the potential mechanism of circRNA in the occurrence and development of ARMs.Then,14 significantly differentially expressed circrnas were selected for expression verification by q RT-PCR.Agarose gel electrophoresis and Sanger clone sequencing were used to detect the base sequence and confirm the circular sites of the products.2.Part Ⅱ: 30 Wistar rats were randomly selected as the experimental group and the ARM group,respectively.The model establishment and sampling method were the same as before.In animal tissue experiments,the expression levels of circJag1,miR-137-3p and Sox9 in fetal rat hindgut tissues were detected by q RT-PCR,and the protein expression level of Sox9 was detected by Western blotting.In order to more clearly display the morphological changes of hindgut tissue development over time in normal and ARMs fetal rats,paraffin-embedded rat embryo tissues were continuously sectionalized in midsagittal plane,and the expression of Sox9 positive cells was detected by immunohistochemical method.In order to simultaneously locate the location distribution of circJag1,miR-137-3p and Sox9 positive cells,fluorescence in situ hybridization(FISH)and immunofluorescence were used for multiple fluorescence co-location detection.In cell experiments,double luciferase plasmids were constructed,co-transfected with Sox9 wild-type and mutant plasmids in the tool cell 293 T,respectively.Circ Jag1wild-type/mutant plasmid and miR-137-3p mimics/negative as control.The luciferase activity ratio of firefly and Renilla Luciferinwas detected to confirm the direct targeted binding site between circJag1 and miR-137-3p/miR-137-3p and Sox9.Linear RNA removal by Rnase R,genomic DNA extraction,agarose gel electrophoresis and Sanger sequencing were used to verify divergent primers and the cyclicity of circJag1.Cell expression location of circJag1 was determined by nuclear and mass separation tests and cell FISH staining.Rat intestinal epithelial cells(IEC-6)were used for RNA Immunoprecipitation(RIP)test to obtain Ago2 protein antibody precipitation corresponding RNA-protein complex.q RT-PCR was used to verify the efficiency of circJag1 bound to the complex,and Sanger sequencing was usedfordetected the product.Results: 1.Part I: A total of 18295 circrnas were identified in the normal and ARM groups.Based on 425 differentially expressed circRNA(|Fc| > 2,p < 0.05),the use of miREAP,miRanda,Target Scan forecasts a total of 55 circRNA(compared with normal group,the ARM grouphas 14 up-regulated,41 down-regulated),195 miRNA and 947 m RNAs were predicted to have potential ce RNA mechanisms.GO and KEGG analysis showed that a large number of differentially expressed circRNAs were enriched in development-related pathways,such as Wnt and MAPK pathways.Only circJag1(novel＿circ＿0011174,derived from the 3-6 exon of Jag1 gene)was significantly differentially expressed in both GD14 and GD15,which was selected as the object of next study.2.Part Ⅱ: The expressions of circJag1 and Sox9 were significantly up-regulated and miR-137-3p was significantly down-regulated in the hindgut tissues of fetal mice in the ARM group.Immunohistochemistry,immunofluorescence and FISH staining results of gross specimens showed that the expression of circJag1 and Sox9 in urorectal septum and cloacal membrane in hindgut tissue of ARM group was significantly higher than that of normal group,indicating that circJag1 and Sox9 of ARM group had temporal and spatial imbalance in the development of fetal hindgut.The results of double lucifase assay showed that the fluorescence ratio was significantly reduced after co-transfection of Sox9 wild-type plasmid and miR-137-3p mimics /circJag1 and miR-137-3p mimics(p<0.01),and the targeted binding site was identified.The products were detected with divergent primers and verified to pass through the cyclization sites.The results of nucleus,mass separation and FISH staining showed that circJag1 was mainly expressed in cytoplasm.RIP assay showed that circJag1 was abundant in Ago2 protein antibody precipitation.In terms of mechanism,through a series of overexpression and interference experiments on circJag1 and miR-137-3p,it was proved that circJag1 had the same expression trend with Sox9,and circJag1 had the opposite expression trend with miR-137-3p.As the molecular sponge of miR-137-3p,circJag1 inhibited the down-regulation of miR-137-3p on its target gene Sox9,and then caused the down-regulation of the expression of β-catenin,cyclin D1 and c-myc in the nucleus.miR-137-3p inhibited circJag1 caused Sox9 up-regulation in a concentration-dependent manner.Cellular immunofluorescence showed that overexpression of circJag1/Sox9 or silencing of miR-137-3p could promote the degradation of β-catenin in the nucleus.Through a series of proliferation and apoptosis tests,it was proved that the high expression of circJag1/low expression of miR-137-3p/high expression of Sox9 promoted the apoptosis and inhibited the proliferation of intestinal epithelial cells.The increased apoptosis and decreased proliferation induced by overexpression of Sox9 could be reversed by transfection of miR-137-3p mimics.Overexpression of miR-137-3p attenuated the pro-apoptotic effect of circJag1,while the loss of Sox9 eliminated the increased apoptosis and decreased proliferation induced by circJag1.Conclusion: 1.Compared to the normal group,there are 425 differentially expressed circRNAs in malformed anorectal tissues of rats,and the malregulated expression of these circRNAs may be an important cause of the occurrence of ARMs.2.The abnormal expression of circJag1 may be closely related to the occurrence of ARMs.Circ Jag1 up-regulation can cause excessive apoptosis of intestinal epithelial cells and inhibit cell proliferation.3.Circ Jag1,as the "sponge" of miR-137-3p,promotes cell apoptosis and inhibits proliferation through miR-137-3p/Sox9 axis,and the pro-apoptotic effect of circJag1 will be abolished due to the loss of Sox9.4.Circ Jag1/Sox9 up-regulation and miR-137-3p down-regulation could lead to the decrease of intracellular β-catenin,thus inhibiting the conduction of Wnt signaling pathway and leading to the down-regulation of cyclin D1 and c-myc expression,which may be an important cause of anorectal malformations. |
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