| Objective:Anorectal malformations(ARMs)are common gastrointestinal malformation in children,with an incidence of about 1/5000 to 1/1500,and it is about 1% higher in patients with family history.ARMs are a group of intestinal malformations with different phenotypes caused by abnormal development of the hindgut during embryonic 4 to 8weeks.The polymorphism of gene mutations causes ARMs to often occur in syndromes.About 60 % of ARMs children have multiple organ system abnormalities,such as urinary,reproductive,cardiovascular,and skeletal,which seriously affect the long-term life quality of children.Recent studies have found that in addition to the involvement of abnormally expressed genes in the pathogenesis of ARMs,ncRNA can also be involved in various aspects of gene expression as well as cell differentiation and organ formation.Although previous studies have found that ncRNA plays a role in anorectal development,the mechanism of lncRNA in anorectal malformations is still unclear and needs to be further studied.At present,the pathogenesis of ARMs is mainly studied through animal models.The rat ARMs induced by ETU gavage is the most commonly used animal model.The key time point of anorectal development in rat embryo is 14-16 days,which corresponds to the time point of anorectal development in human embryo from 4 to 8 weeks.In this study,we performed whole transcriptome sequencing on the hind gut tissues of six normal groups and teratogenic ETU rat at GD14,GD15 and GD16.Combined with bioinformatics analysis,we screened differentially expressed genes and ncrnas and analyzed their association.To explore the key lncrnas and genes related to development at the animal,cellular and molecular levels,and to clarify the lncrnas and their related molecular mechanisms that play important roles in the occurrence of ARMs.Methods: 1.Preparation of anorectal malformation animal model and specimen collection.The animals were sexually mature Wistar rats,which were mated at night,and vaginal smears were taken in the morning of the next day,and the sperm was observed under the microscope to be day 0 of pregnancy(GD0).Rats in ARMs group were treated with 1% ETU12.5ml/kg by gavage on GD10.The normal group was given normal saline by gavage.The fetuses were delivered by cesarean section at GD14,GD15,and GD16.Some embryos were immediately removed from the hindgut tissue to extract primary cells or quickly stored in liquid nitrogen for subsequent experiments,and some embryos were fixed in paraformaldehyde and embedded in paraffin.2.Differentially expressed mRNAs and ncRNAs in the hindgut of ARMs and normal fetal rats by whole transcriptome sequencing.The GD14-GD16 hindgut tissues of the normal group and ARMs group were taken for whole transcriptome sequencing(Guangdong gene denovo Biotechnology Co.,LTD.).First,differential mRNAs and lncRNAs were screened out,and then Weighted Gene Co-Expression Network Analysis(WGCNA)was used to analyze the gene modules related to hindgut development.Furthermore,GO and KEGG functional enrichment analysis were performed based on the developmental related gene modules to understand the possible biological functions and signaling pathways of these genes.Finally,lncRNA-target gene-signaling pathway network diagram was constructed through the association analysis of lncRNA and genes to understand the biological functions of lncRNA.Important genes and lncRNAs were validated by RT-qPCR.3.To study lnc53086 and Pax1 as research indicators,to verify the temporal and spatial expression of lnc53086 and Pax1 in normal and ARMs fetal intestinal tissues by FISH and IHC,and to study the effects of lnc53086 and Pax1 on the proliferation of fetal intestinal epithelial cells by CCK8,Ed U and flow cytometry.The role of cell cycle and apoptosis.We verified that lnc53086 and Pax1 bound to the protein by RNA pulldown and RIP experiments.According to the binding sites of mi R-0204-3p to lnc53086 and Pax1,dual luciferase reporter plasmids were designed and synthesized.We confirmed whether mi R-0204-3p directly bound to lnc53086/Pax1 by co-transfection of the plasmid with mi R-0204-3p mimics/NC,and the changes of renia fluorescence and firefly fluorescence were detected by microplate reader,respectively.After knocking down lnc53086/mi R-0204-3p or overexpressing mi R-0204-3p,the expression of target gene Pax1 was detected.4.Transcriptional regulation of Pax1 on target genes and its effect on Wnt/β-catenin pathway.After overexpression of FLAG-tagged Pax1 adenovirus in rat intestinal epithelial cells,CHIP-seq(chromatin immunoprecipitation)mined the binding sites of Pax1 in the promoters of downstream genes,and the downstream genes related to ARMs were found by association analysis with the transcriptome.CHIP-qPCR was used to verify the related genes,and the expression of downstream genes was observed after overexpression of Pax1.CO-IP(co-immunoprecipitation)was used to verify the binding of Pax1 to Tcf4,and Western blot was used to verify the effect of Pax1 on the protein expression of β-catenin.5.The experimental data were statistically analyzed using Graph Pad Prism 8.0,and graphs were generated.Independent sample t test was used to compare the differences between the two groups,and one-way analysis of variance(ANOVA)was used to compare the statistical differences between more than three groups of data.P < 0.05 was considered to indicate statistical significance.Results: 1.A total of 4184 lncRNAs and 39713 mRNAs transcripts were reconstructed by whole transcriptome sequencing.A total of 314 significantly differentially expressed lncRNAs and 3546 significantly differentially expressed mRNAs were identified in the 6groups.Compared with the normal group,there were 31 differentially expressed lncRNAs and 431 mRNAs in the anorectal malformation group.By WGCNA analysis,it was found that the differentially expressed mRNAs were mainly located in the blue module,and the blue module genes were involved in metabolic process,developmental process,cell component organization,MAPK signaling pathway,PI3K-Akt signaling pathway,focal adhesion and Wnt signaling pathway.By lncRNA-mRNA co-expression analysis,we found that 89 differentially expressed lncRNAs were co-expressed with 97 differentially expressed mRNAs.The most important pair is lnc53086-Pax1,which is regulated by Trans analysis and has a similar expression pattern,and they are highly expressed in ARMs groups at GD14 and GD15.Functional analysis showed that these genes were related to anatomical structure development,developmental processes and multicellular biological processes,and were also involved in MAPK signaling pathway,Hippo signaling pathway and Wnt signaling pathway.Finally,we selected 5 lncRNAs and 5 mRNAs for RT-qPCR verification,and the results were consistent with the sequencing results.2.Fluorescence in situ hybridization showed that lnc53086 and Pax1 were highly expressed in the ARMs,and they were mainly distributed in the distal hindgut and cloacal membrane.The results of immunohistochemistry and Western Blot showed that Pax1 was up-regulated in ARMs.Knockdown of lnc53086 can promote the cycle of rat intestinal epithelial cells(IEC),increase proliferation,and reduce apoptosis.Overexpression of Pax1 resulted in opposite results.3.RNA pulldown of lnc53086 showed that lnc53086 could bind to AGO2.Further RIP experiments showed that AGO2 could bind to lnc53086 and Pax1.After co-transfection of lnc53086/Pax1 3 ’-UTR wild-type plasmid and mi R-0204-3p mimics,renilla/firefly luciferase activity was significantly decreased.After knocking down lnc53086,RT-qPCR confirmed that the expression level of Pax1 was decreased.4.By Chip-Seq and RNA-seq association analysis,we found that Pax1 bound to 84 differential gene promoters.These genes are mainly involved in mismatch repair,DNA replication nucleotide excision repair pathways,cell developmental processes,cell differentiation and animal organ development.Eight genes with significant differences(myc,six1,fn1,cdh6,lgals1,rock1,gatad2 b,n1fx)were selected for verification.Chip-PCR confirmed that Pax1 could bind to the promoters of these genes,and the expression of these genes was decreased after transfection of overexpressing Pax1 adenovirus.Most of these genes are the target genes of Wnt/β-catenin pathway,and the expression of β-catenin protein was decreased after overexpression of Pax1.By COIP and immunofluorescence,we demonstrated that Pax1 could bind to Tcf4,a Wnt/β-catenin downstream transcription factor.Conclusions:1.Whole transcriptome sequencing results suggest that there are many different ncRNAs and mRNAs in the hindgut tissues of normal and ARMs fetal rats.Among them,lncRNA is involved in developmental related signaling pathways by interacting with protein coding genes,and the expression of lnc53086 and its target gene Pax1 is significantly different and has a consistent trend,and is related to the signaling pathway related to ARMs,suggesting that lnc53086 and Pax1 may be relatively key molecules affecting ARMs.2.Lnc53086 and Pax1 were highly expressed in ARMs and mainly distributed in the distal hindgut and cloacal membrane.lnc53086 can promote the expression of Pax1 by acting as a molecular sponge for mi R-0204-3p through ceRNA mechanism,and induce the proliferation inhibition and apoptosis of IEC by mediating Pax1,thereby leading to the ARMs phenotype.3.Pax1 can directly bind to Tcf4 and participate in Wnt/β-catenin signaling pathway.Pax1 acts as a transcriptional repressor to inhibit β-catenin nuclear translocation,thereby inhibiting the expression of the downstream genes of Tcf4.Overexpression of Pax1 can decrease the expression of β-catenin and down-regulate the downstream genes.In summary,the lnc53086/Pax1/Tcf4 signaling axis may be a pathway for lnc53086 to exert its biological function. |
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