| Objective:Anorectal malformations（ARMs）is one of the most common congenital malformations of the digestive system in children.It usually manifests as anal atresia,ectopic anus,urethral rectal fistula,etc.The incidence of this disease is about 1/50001/1500.ARMs is usually associatted with malformations of other system.Most patients with ARMs need surgery.However,long-term follow-up results show that many ARMs have post-operative complications such as constipation and incontinence,which seriously affects the quality of life and mental health.Therefore,it is of great significance to have a deep understanding of the pathogenesis of ARMs and to provide better treatments based on this.During the normal anorectal development,the fusion of urogenital septum and cloacal membrane,the complete separation of rectum and urethra,and the complete opening of terminal rectum are essential for the formation of the anus.When this process is blocked by external factors,dysplasia may occur and anorectal malformations may happen.Because clinical specimens are difficult to obtain,experimental animal models are generally used for the research of ARMs.Among them,ETU（ethylenethiourea）-induced rat embryos with anorectal malformation are the most commonly used animal models.Morphological studies have found that the major phenotypes of ARM malformed embryos include:（1）fusion failure of urethral rectal septum and cloacal membrane;（2）delayed tailgut degression;（3）abnormal cloacal wall apoptosis;（4）underdevelopment of dorsal cloaca and dorsal cloacal membrane.In this process,GD15 and GD16 are important time points for the fusion of urogenital septum and cloacal membrane and anus formation.If the anus is not present or there is still a passage between the urethra and the rectum on GD16,anorectal malformations will inevitably occur.The etiology of ARMs is complex,and various genetic and environmental factors may cause disease phenotypes.Previous studies have shown that abnormal expression of multiple genes may be involved in the pathogenesis of ARMs,and the most studied signaling pathways are Wnt,Notch,Shh,and TGF-β.However,the specific mechanism of ARMs is still unclear and needs to be studied in depth.The miRNA（microRNA）discovered in recent years can regulate target genes at post-transcriptional level by binding to the 3’-UTR of the mRNAs.It has been reported that miRNAs have important effects in the development of various congenital diseases and embryonic development in multiple systems.Previous studies have suggested that miRNAs play a role in the development of digestive tracts in various species,and miRNA abnormalities can cause a wide range of digestive tract diseases.However,current understanding of the miRNA function in the development of ARMs is still limited.Comparing miRNA expression profiles between normal rats and rats with anorectal malformations can help to reveal the role of miRNAs in the development of ARMs,providing a valuable basis for clarifying the pathogenesis of anorectal malformations.In this study,we selected fetal rats at critical periods of anorectal development（i.e.,GD14-GD16）to profile miRNA expression patterns in the hindgut of normal and ETU-induced fetuses with anorectal malformations,with subsequent biological information analyses,aiming to find out differentially expressed miRNAs.The key miRNAs were then explored at the animal,cell,and molecular levels to identify relevant mechanisms that may play an important role in the development of ARMs.Methods:1.Preparation and specimen collection of anorectal malformations animal model.The animals used were 7-9 weeks old,250-300g body weight,and sexually mature Wistar rats,which were provided by Experimental Animal Center of Shengjing Hospital affiliated to China Medical University and raised in an SPF animal breeding room（room temperature 22±2°C,humidity 55±5°C,maintaining 12 hours of day and night cycle）.Male rats were put together with female rats at a ratio of 1:5 at midnight,and the vaginal smear was made to make preliminary judgment of pregnancy in the morning of the next day.The day when sperms were found under the light microscope was designated as the first day of pregnancy（Gestational day 0,GD0）.Pregnant rats were randomly assigned to the ETU treatment group（ARMs group）and the normal control group（Normal group）.The ARMs group was gavaged with1%ETU at a dose of 12.5ml/kg on GD10,while the normal group was gavaged with corresponding normal saline.Continue to raise the rats after gavage.Till GD14,GD15,or GD16,take the fetuses by cesarean section.Some of the embryos were fixed in 4%paraformaldehyde,dehydrated in graded alcohol,paraffin embedded in xylene,and sliced in 4μm for immunohistochemistry,fluorescently labeled TUNEL staining,and in situ hybridization,while hindgut tissues of the other embryos were taken out and quickly stored in liquid nitrogen for subsequent RT-qPCR,Western Blotting or other experiments.2.The miRNAs differentially expressed in the hindgut tissues of normal and ARMs fetuses were screened.The miRNA chip was designed based on miRBase database,and the hindgut tissues of normal and ARMs group were taken for miRNA chip detection（Guangdong Ruibo Biotechnology Co.,Ltd.）.After screening for differentially expressed miRNAs,the target genes were predicted miRNAs with larger change folds.Then,based on the target genes,GO and KEGG functional enrichment analysis were performed to investigate relevant biological functions and possible signaling pathways.Regulatory networks of miRNA-target genes and miRNA-target genes-related signaling pathways were subsequently constructed.Core miRNAs were validated using RT-qPCR.3.Confirmation of the targeting relationship between miR-92a-2-5p and PRKCA,and their spatio-temporal expression patterns in normal and ARMs hindgut tissues.After predicting the binding site of miR-92a-2-5p on PRKCA by TargetScan,we designed and constructed a dual luciferase reporter plasmid containing the renilla luciferase gene,the firefly luciferase gene,PRKCA 3’-UTR（wild type,wt）with normal binding site or PRKCA 3’-UTR（mutant type,mut）with mutated binding site,and the plasmid is co-transfected with miR-92a-2-5p mimics or its negative control.The activity of renilla luciferase and firefly luciferase were examined to explore whether there is a direct targeting relationship between miR-92a-2-5p and PRKCA.For the spatio-temporal expression of miR-92a-2-5p in normal and ARMs,we used fluorescence in situ hybridization assay.As for its target gene PRKCA,Western Blotting was used to detect protein expression and IHC was used to detect PRKCA positive cell distribution.4.The effect of miR-92a-2-5p on the expression of target gene PRKCA and downstreamβ-catenin.Experiments were performed using rat intestinal epithelial cells.After overexpression or knockdown of miR-92a-2-5p,RT-qPCR was used to detect the transfection efficiency and determine the optimal transfection concentration.Three siRNAs were designed for PRKCA,and the transfection efficiency was detected by RT-qPCR.The siRNA with best silencing effect was subjected to subsequent experiments.After overexpressing miR-92a-2-5p or silencing PRKCA,Western Blotting was used to detect the expression levels of PRKCA andβ-catenin.5.The effect of miR-92a-2-5p and PRKCA on the function of rat intestinal epithelial cells.Cell cycle,cell proliferation,and caspase 3/7-related apoptosis were detected after overexpressing miR-92a-2-5p or silencing PRKCA,and changes in the proportion of early/late apoptotic cells were observed using flow cytometry.The paraffin sections were stained with TUNEL to investigate the strength and spatial distribution of apoptotic signals in normal and post-intestinal tissues of ARMs.6.Statistical analysis.Experimental data were expressed as mean±standard deviation and statistical analysis was performed using SPSS 21.0 software（IBM SPSS,USA）.For the comparison between the two groups,Student’s t-test（two-tailed）test was used;and for comparison between multiple groups,one-way ANOVA and Tukey post-hoc analysis were used.Statistical significance was considered significant with a standard of P<0.05.Results:1.With absolute value of log2（Fold change）>1 and P value<0.05 as the standard of differentially expressed miRNAs in the chip,we found that on GD14,38 miRNAs were up-regulated in ARMs group compared with the normal group;on GD15,32miRNAs were up-regulated and 17 down-regulated;and on GD16,42 miRNAs were up-regulated.RT-qPCR was performed for miRNAs with|log2（FC）|>4.25 and potentially associatted with ARMs-related signaling pathways,and results showed that miR-125-2-3p,miR-92a-2-5p,and miR-99a-5p were most significantly and differentially expressed.Among them,miR-92a-2-5p was most widely associated with target genes and signaling pathways in the miRNA-target gene-signal pathway network.2.In the dual luciferase assay,renilla/firefly luciferase activity was significantly decreased（P<0.05）when co-transfecting PRKCA 3’-UTR wt with miR-92a-2-5p mimics.The fluorescence in situ hybridization results of miR-92a-2-5p showed that miR-92a-2-5p was highly expressed in the ARMs group,and mainly distributed in the terminal rectum and cloacal membrane.Immunohistochemistry and Western Blotting results showed that PRKCA was down-regulated in ARMs.3.After overexpressing miR-92a-2-5p or silencing PRKCA,the protein expression level of PRKCA was decreased,while the protein expression level ofβ-catenin was increased.Cellular immunofluorescence results showed that miR-92a-2-5p overexpression or PRKCA knockdown could lead to a change in the subcellular localization ofβ-catenin,causing it to accumulate in the nucleus.4.Overexpression of miR-92a-2-5p increased cell death in a dose-dependent manner.The higher the concentration of miR-92a-2-5p,the more significant the cell death was.The results of fluorescent labeling of paraffin sections TUNEL showed that the apoptotic signal was enhanced in the posterior intestine of ARMs,and the distribution of apoptotic cells was similar to that of fluorescent in situ hybridization in miR-92a-2-5p.5.Overexpression of miR-92a-2-5p can cause G1/S phase arrest,proliferation deduction and apoptosis induction in rat intestinal epithelial cells.Apoptotic cells were classified into late apoptosis,while the proportion of early-stage apoptosis was also increased a little.Similar results were observed after silencing PRKCA.Conclusion:1.The results of the microarray suggest that there were many differentially expressed miRNAs in the hindgut tissues between normal and ARMs fetal rats,and the miRNA upregulation in ARMs group is dominant.These differentially expressed miRNAs have potential links with development-related signaling pathways,and miR-92a-2-5p exhibited the most extensive connections with target genes and ARMs-related signaling pathways,suggestting that miR-92a-2-5p may be a relatively critical miRNA in ARMs.2.miR-92a-2-5p can directly target PRKCA,and their expression trends in normal and ARMs hindgut were opposite.Overexpressing miR-92a-2-5p or silencing PRKCA resulted in down-regulation of PRKCA and up-regulation ofβ-catenin,suggesting that miR-92a-2-5p/PRKCA/β-catenin signaling axis may be the way miR-92a-2-5p function in ARMs.3.miR-92a-2-5p was increased in ARMs,mainly distributed in the terminal hindgut end and cloacal membrane.The distribution of TUNEL signals were similar to miR-92a-2-5p.After overexpressing miR-92a-2-5p or silencing PRKCA,intestinal epithelial cell death was increased,cell proliferation was deduced,and cell apoptosis was enhanced,suggesting that miR-92a-2-5p can mediate the inhibition of proliferation and promotion of apoptosis in intestinal epithelial cells via PRKCA,which may lead to the phenotype of ARMs phenotype. |
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