MiR-9a-5p Participates In The Pathogenesis Of Anorectal Malformation In Rats By Targeting CBX6 To Affect The Wnt/β-catenin Signal Pathway

Objective: Congenital anorectal malformation is the most common digestive tract malformation.Postoperative defecation dysfunction seriously affects the quality of life of children,and its pathogenesis is unclear at present.Studies have shown that microRNA is closely related to the occurrence of congenital anorectal malformation.Based on the animal model of anorectal malformation in rats induced by ETU,this study is to explore the possible role of miR-9a-5p in the pathogenesis of anorectal malformation.Methods: 1.The difference analysis of transcriptome miRNA data was carried out using R language pheatmap package and Gideo Bioinformatics cloud tool,and the key differential miRNA was verified by qRT-PCR.2.The sexually mature Wistar rats were selected for caging,and the pregnant rats after caging were randomly divided into normal group and ARM group;Pregnant rats in GD10 normal group were gavaged with normal saline according to 125mg/kg standard,and pregnant rats in ARM group were gavaged with 1% ETU according to 125mg/kg standard;The anorectal tissues of fetal rats were collected at GD14,GD15 and GD16.3.The spatiotemporal expression of miR-9a-5p in GD14,GD15,GD16 normal group and ARM group GD14,GD15,GD16 was observed by FISH.4.The target gene of rno-miR-9a-5p was predicted by mir Walk,miRDB and Target Scan,and the direct targeting relationship between miR-9a-5p and CBX6 was verified by double luciferase.5.The expression of CBX6 mRNA and protein was detected by qRT-PCR,Western blot and immunohistochemical staining.6.After overexpression of miR-9a-5p in IEC-6 cells,the transfection efficiency,CBX6 mRNA and protein β-Catenin protein expression,IEC-6 cell proliferation and apoptosis were detected by CCK-8,Annexin Ⅴ-FITC/PI.7.After knocking down CBX6 in IEC-6 cells,the transfection efficiency was detected by qRT-PCR,the expression of CBX6 andβ-catenin protein was detected by Western blot,and the proliferation and apoptosis of IEC-6 cells were detected by CCK-8,Annexin Ⅴ-FITC/PI.8.SPSS19.0 and Prism8.0 software were used for statistical analysis.All experimental data were expressed with mean ± standard deviation.The statistical data were repeated at least three times,and P<0.05 indicated that the difference was statistically significant.Results: 1.MiR-9a-5p was significantly up-regulated in the hindgut of GD14,GD15 and GD16 ARM fetal rats.2.FISH showed that miR-9a-5p in the normal group mainly detected fluorescence signals along the urethra and intestines,while in the ARM group,miR-9a-5p could detect extensive and obvious fluorescence signals in the urethra,urethrorectal septum,rectourethral fistula,cloacal membrane and intestines.3.The double luciferase experiment confirmed that miR-9a-5p can directly target the 3 ’UTR region of CBX6.4.The expression levels of CBX6 mRNA and protein were downregulated in the hindgut tissues of GD14,GD15 and GD16 ARM fetal rats.5.Immunohistochemical staining showed that CBX6 was widely and strongly expressed in the urethra,urethrorectal septum,rectourethral fistula,cloacal membrane and rectum of the normal group,while CBX6 was weakly expressed in the urethra and rectum of the ARM group.6.Overexpression of miR-9a-5p will down-regulate the expression of CBX6 mRNA and protein,β-Catenin protein expression is down-regulated,which inhibits cell proliferation and promotes cell apoptosis.7.Knock down BX6 will downregulate the expression of CBX6 and β-catenin protein,inhibit cell proliferation and promote cell apoptosis.Conclusion: The results show that miR-9a-5p can target CBX6 to affect Wnt/ β-Catenin signal pathway plays a role in the occurrence and development of ARMs.