| Objective:Congenital anorectal malformation is one of the common malformations in pediatric surgery and most of them can be diagnosed in the neonatal period.Although children with anorectal malformation can be treated surgically,but about50%of children combined with urogenital system,cardiovascular system,skeletal deformity and other system deformities,and the prognosis effect is poor.During the embryonic development,the genes which are related to midline organ fusion,cell adhesion fusion and caudal ectoderm are often abnormal exresssion in anorectal malformation.The Wnt signaling pathway is one of the most mature and important pathways in the study of anorectal malformation,but but it is not clear how the Wnt signaling pathway upstream regulates the development of anorectal malformation.Micro RNA?miRNA?can inhibit the Wnt gene expression process by binding to the 3'non-coding region of the Wnt gene.Circular RNA?circRNA?is considered to be the most competitive mi RNA"sponge"due to its strong stability and high conservatism to regulate the expression of target genes by competitively binding to the 3'non-coding region of mRNA.CircRNAs play an important role in gliomas,breast cancer,lung cancer,liver cancer,gastric cancer and other diseases,but there are still few studies on anorectal malformations.How does circRNA act on miRNA and then regulate Wnt signaling pathways on the development of anorectal malformations is still worth exploring.Methods:1.Female?250–280 g?and male?280–300 g?Wistar rats were provided by the Chang Sheng Biotechnology.Pregnant rats were identified by sperm in vaginal smears after overnight mating,and the day was defined E0.A total of 84 pregnant Wistar rats were randomly divided into two groups:ETU?2-imidazolidinethione?-induced ARM and control groups?42 pregnant rats were administered a single dose of ETU and 42 pregnant rats were administered an equal dose of saline without ETU via oral gavage on E10?.The concentration of ETU was 0.01 mg/mL and the dose was125 mg/kg.Embryos were harvested by cesarean delivery on E16,E17,E19,and E21.The embryos were divided into ARM?short or no tail?and control?long tail?groups.The cloacae and hindguts were dissected,removed from surrounding tissues under magnification,and immediately frozen in liquid nitrogen.Long-tailed fetuses were classified as the control group,and some embryos were placed in 4%polyformaldehyde for 48-72 hours,then embedded paraffin.The remaining embryos were collected from the anorectal malformation group and the control group of fetal rats in the uretorectal septum,cloaca and hindgut region tissues.They were placed in liquid nitrogen and stored at-80°C in a refrigerator.2.E16 and E17 tissues in the deformity group and the control group were subjected to high-throughput sequencing.Candidate circRNA were screened out based on the differential expression multiples and KEGG analysis.qRT-PCR was used to verify the candidate circRNA between the ARM group and the normal control group.Sanger sequencing was used to determine the full-length sequence of rno?circ?0005139 and the circularization site.Lipo3000was used to transfect the rno?circ?0005139 si RNA and overexpression plasmid in IECs cell line?rat intestinal epithelial cell line?.The transfected cells were then subjected to cell proliferation and apoptosis.And qRT-PCR experiments to verify the function of rno?circ?0005139 and mRNA level after transfection.3.According to miRanda and psRobot software,predict miRNA which were with the binding site of rno?circ?0005139,and predict mi RNA with binding sites to Wnt5a/Wnt3a by targetscan.The anorectal tissues of the E17-21 in ARM group and normal control group were to verify the expression of miR-324-3p/mi R-128-3p by qRT-PCR.A dual luciferase assay was used to identify the binding sites between rno?circ?0005139an miR-324-3p/miR-128-3p and the binding sites between miR-324-3p/miR-128-3p and Wnt5a/Wnt3a.The co-transfected rno?circ?0005139 siRNA and miR-324-3p/miR-128-3p,inhibitor or the overexpression plasmid of rno?circ?0005139 and miR-324-3p/miR-128-3p mimics were used to perform cell proliferation,apoptosis,and to verify Wnt5a/Wnt3a m RNA and protein levels by qRT-PCR and Western blot.4.Statistical analysis.Set at least 3 biological replicates in each group.The relative density of the bands was detected by Image-J software for Western blot analysis.The qRT-PCR data were expressed by 2-??Ctvalues.Statistics were performed using SPSS 21.0 statistical software.For the data that obey the normal distribution,the two-sample t test is used for comparison between the experimental group and the control group.For the data that does not obey the normal distribution,the Mann-Whitney U test is used for the comparison between the experimental groups,All experimental data are expressed as mean±standard deviation,p<0.05 is considered statistically significant.Results:1.CircRNA sequencing and verification in anorectal tissues of ARM group and normal control group.According to the sequencing of circRNAs,16 circRNAs were up-regulated,40 circRNAs were down-regulated at E16 between the ARM group and the normal control group.And 38 circRNAs were up-regulated at E17,and42 circRNAs were down-regulated?|Fold Change|>2 and P<0.05?.According to the fold change and KEGG analysis,we selected the following six circRNAs as candidate?rno?circ?0000436,rno?circ?0005139,rno?circ?0006880,rno?circ?0009285,rno?circ?0011909,rno?circ?0014367?,and verified their expression by qRT-PCR.Finally,the expression of rno?circ?0006880 was up-regulated in the ARM group,and rno?circ?0000436,rno?circ?0005139,rno?circ?0009285,rno?circ?0011909,and rno?circ?0014367 are down-regulated in the ARM group.Finally,we selected rno?circ?0005139 as the final candidate circRNA because of the expression trend in anorectal tissue and its targeting miRNAs which can bind with Wnt5a/Wnt3a.2.Rno?circ?0005139 identification,cyclization site determination and functional verification.The full length of rno?circ?0005139was 1301bp.Rno?circ?0005139 was mainly present in the cytoplasm by nuclear-plasma separation test,and rno?circ?0005139 siRNA in IECs cells promoted apoptosis and inhibited proliferation.3.The mechanism and function of rno?circ?0005139-miR-324-3p-Wnt5a,and rno?circ?0005139-miR-128-3p-Wnt3a pathway in ARM group and normal control group.According to miRanda,psRobot and Targetscan prediction software,miR-324-3p and miR-128-3p were predicted to targete rno?circ?0005139 and the Wnt5a was with binding sites of miR-324-3p and Wnt3a was with binding sites of miR-128-3p.And the double luciferase experiments has confirmed that there were binding sites between rno?circ?0005139 and miR-324-3p/miR-128-3p,between miR-324-3p and Wnt5a,and between miR-128-3p and Wnt3a.And miR-324-3p and miR-128-3p were up-regulated in ARM group by qRT-PCR,and Wnt3a and Wnt5a were down-regulated in ARM group.It can be confirmed that rno?circ?0005139 can be used as miR-324-3p and mi R-128-3p sponges target Wnt5a and Wnt3a to negatively regulate their expression.In addition,miR-324-3p inhibitor and miR-128-3p inhibitor reversed the effects of rno?circ?0005139 siRNA on Wnt5a and Wnt3a expression,the degree of apoptosis.Therefore,we conclude that rno?circ?0005139/miR-324-3p/Wnt5a and rno?circ?0005139/miR-128-3p/Wnt3a play a vital role in ARM,and cell function of apoptosis.Conclutions:1.CircRNA existed in rat anorectal tissues,and they are obvious differences between ARM group and normal control group.2.Rno?circ?0005139participated in the development of anorectum.Rno?circ?0005139 was reduced in ARM group and inhibited apoptosis.3.Rno?circ?0005139/miR-324-3p/Wnt5a and rno?circ?0005139/miR-128-3p/Wnt3a were involved in the process of anorectal malformation,affecting cell proliferation and apoptosis.Rno?circ?0005139 may be a potential therapeutic target in anorectal malformation. |
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